CD138-targeted interferon demonstrates potent apoptotic and anti-tumor activities

ABSTRACT

In various embodiments chimeric moieties (constructs) are provided that show significant efficacy against cancers. In certain embodiments the constructs comprise a targeting moiety that specifically binds CD138 attached to an interferon or to a mutant interferon. In certain embodiments, the constructs comprise anti-CD138 antibody attached to an interferon alpha (IFN-α) or to a mutant interferon alpha.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. Ser. No. 14/649,888, filed Jun.4, 2015, which is a U.S. 371 National Phase of PCT/US2013/073410, filedDec. 5, 2013, which claims priority to and benefit of U.S. Ser. No.61/734,851, filed Dec. 7, 2012, all of which are incorporated herein byreference in its entirety for all purposes.

STATEMENT OF GOVERNMENTAL SUPPORT

[ Not Applicable ]

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file,“UCLA-P097D1US_ST25.txt” created on Feb. 27, 2018 and having a size of25,659 bytes. The contents of the text file are incorporated byreference herein in their entirety.

BACKGROUND

Multiple myeloma (MM) is a disease characterized by an excess ofmalignant plasma cells in the bone marrow (BM). Most MM cells secreteIgG or IgA that contain somatic hypermutations, suggesting that thecritical transformation processes occur in the germinal centers and mayinvolve antigenic stimulation. Accumulation and proliferation ofmalignant myeloma cells result in disruption of normal hematopoiesis andchanges to BM vascularization and bone physiology. MM is the second mostprevalent hematologic malignancy in the US with a survival rate of 7-8years when patients are treated with drugs such as proteasome inhibitorbortezomib, or thalidomide and lenalidomide, which target MM cells inthe BM microenvironment (Kumar et al. (2008) Blood 111: 2516-2520).

MM is characterized by a wide variety of genetic mutations. Analyses oflarge numbers of patient MM cells and human myeloma cell lines (HMCLs;Carrasco et al. (2006) Cancer Cell 9: 313-325; Drexler et al. (2000)Leukemia, 14: 777-782; Lombardi et al. (2006) Genes Chromosomes Cancer,46: 226-238; Moreaux et al. (2011) Haematologica, 96: 574-582) attest tothe molecular heterogeneity of this disease. There are two oncogenicpathways that are responsible for the initial onset of MM or thepremalignant disease called monoclonal gammopathy of undeterminedsignificance (MGUS)—hyperdiploidy sometimes containing multipletrisomies of chromosomes 3, 5, 7, 9, 11, 15, 19, and 21 and primaryimmunoglobulin translocations involving 11q13 (CCND1), 4p16(FGFR3/WHSC1), 6p21 (CCND3), 16q23 (MAF), and 20q11 (MAFB), which resultin dysregulated expression of the target genes. Disease progression ismarked by activating mutations to K- or N-Ras and inactivation ofCDKN2A, CDKN2C, CDKN1B, and/or PTEN tumor suppressor genes. As tumorsbecome more aggressive in later stages of the disease, secondary Igtranslocations involving MYC have been found in MM. This is in contrastto human Burkitt's lymphoma and murine plasmacytoma in which c-myctranslocation is an early oncogenic event. In addition, mutations and/ordeletions of p18 and p53 have been observed as late events in MMpathogenesis.

Interferons have been contemplated for use in the treatment of cancer(Borden et al. (2000) Semin. Cancer Biol., 10: 125-144; Borden et al.(2007) Nat. Rev. Drug Discov., 6: 975-990). There are seven classes oftype I IFNs with IFNα and IFNβ being the most abundant. Both IFNα andIFNβ bind to the same receptor composed of two transmembrane proteins,IFNAR 1 and 2, but IFNβ binds with much higher affinity than IFNα(Lamken et al. (2004) J Mol Biol 341: 303-318). IFNs have been shown tohave anti-proliferative activity as well as the ability to induceapoptosis in hematological malignancies and solid tumors in addition totheir anti-viral activity (as reviewed in Borden et al. (2007) Nat. Rev.Drug Discov., 6: 975-990). However, the effectiveness of IFNα for cancertherapy is overshadowed by side effects when used at high doses (Weiss(1998) Semin. Oncol., 25: 9-13) and by a short half-life of only 1 hour(Peleg-Shulman et al. (2004) J. Med. Chem., 47: 4897-904). Strategies toincrease the half-life have included the covalent linkage ofpolyethylene glycols (PEG) to IFNα (Talpaz et al. (2001) Blood, 98:1708-1713), but such modifications have resulted in lower activity(Rosendahl et al. (2005) Bioconjug. Chem., 16: 200-207).

SUMMARY

In various embodiments this invention pertains to the discovery thatattaching an interferon to a targeting moiety (e.g., a molecule thatspecifically and/or preferentially binds a marker on or associated witha cell) substantially improves the therapeutic efficacy of theinterferon and appears to reduce systemic toxicity. Accordingly, invarious embodiments, this invention provides constructs comprising aninterferon attached to a targeting moiety and uses of such constructs tospecifically and/or preferentially inhibit the growth or proliferationor even to kill certain target cells (e.g., cancer cells). In certainembodiments the constructs comprise a mutant interferon, e.g., a mutantIFNα with higher affinity for the IFNAR to enhance the potency of theconstruct.

Accordingly, in certain embodiments, a chimeric construct is providedwhere the construct comprises an interferon (e.g., interferon-alpha,interferon-beta, interferon-gamma, mutant interferon-α, mutantinterferon-β, and the like) attached to a targeting moiety that binds toa tumor associated antigen, in particular CD138. The construct whencontacted to a tumor cell results in the killing or inhibition of growthor proliferation of the tumor cell.

In various aspects, the invention(s) contemplated herein may include,but need not be limited to, any one or more of the followingembodiments:

Embodiment 1: A construct including an interferon attached to anantibody that binds CD138.

Embodiment 2: The construct of embodiment 1, wherein said construct whencontacted to a cell that expresses or overexpresses CD138 cell resultsin the killing or inhibition of growth or proliferation of said cell.

Embodiment 3: The construct of embodiment 2, wherein said cell thatexpresses or overexpresses CD138 is a cancer cell.

Embodiment 4: The construct of embodiment 2, wherein said cell thatexpresses or overexpresses CD138 is a cancer from a cancer selected fromthe group consisting of multiple myeloma, ovarian carcinoma, cervicalcancer, endometrial cancer, kidney carcinoma, gall bladder carcinoma,transitional cell bladder carcinoma, gastric cancer, prostateadenocarcinoma, breast cancer, prostate cancer, lung cancer, coloncarcinoma, Hodgkin's and non-Hodgkin's lymphoma, chronic lymphocyticleukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloblasticleukemia (AML), a solid tissue sarcoma, colon carcinoma, non-small celllung carcinoma, squamous cell lung carcinoma, colorectal carcinoma,hepato-carcinoma, pancreatic cancer, and head and neck carcinoma.

Embodiment 5: The construct according to any one of embodiments 1-4,wherein said interferon is a type I interferon.

Embodiment 6: The construct according to any one of embodiments 1-4,wherein said interferon is a type II interferon (IFNγ).

Embodiment 7: The construct of embodiment 5, wherein said interferon isan interferon-alpha (e.g., IFNα2).

Embodiment 8: The construct of embodiment 7, wherein said interferon isan interferon alpha 2 (IFNα2).

Embodiment 9: The construct of embodiment 7, wherein said interferon isan interferon alpha 14 (IFNα14).

Embodiment 10: The construct of embodiment 5, wherein said interferon isan interferon-beta (IFNβ).

Embodiment 11: The construct according to any one of embodiments 7-10,wherein said interferon is a human interferon.

Embodiment 12: The construct according to any one of embodiments 7-10,wherein said interferon is a non-human interferon.

Embodiment 13: The construct of embodiment 12, wherein said interferonis a murine interferon.

Embodiment 14: The construct according to any one of embodiments 1-4,wherein said interferon is a mutant type I interferon.

Embodiment 15: The construct of embodiment 14, wherein said interferonis a mutant interferon-alpha.

Embodiment 16: The construct of embodiment 14, wherein said interferonis a mutant human interferonα-2 having mutations at one or more sitesselected from the group consisting of His57, Glu58, and Gln61.

Embodiment 17: The construct of embodiment 16, wherein said interferonis an interferonα-2 having a mutation at His57.

Embodiment 18: The construct of embodiment 17, wherein said mutation atHis57 is a mutation to an amino acid selected from the group consistingof A, Y, and M.

Embodiment 19: The construct according to any one of embodiments 16-19,wherein said interferon is an interferonα-2 having a mutation at Glu58.

Embodiment 20: The construct of embodiment 19, wherein said mutation atGlu58 is a mutation to an amino acid selected from the group consistingof A, N, D, and L.

Embodiment 21: The construct according to any one of embodiments 16-20,wherein said interferon is an interferonα-2 having a mutation at Gln61.

Embodiment 22: The construct of embodiment 19, wherein said mutation atGln61 is a mutation to an amino acid selected from the group consistingof A, S, and D.

Embodiment 23: The construct of embodiment 16, wherein said interferonincludes the mutations H57Y, E58N, and Q61S.

Embodiment 24: The construct of embodiment 16, wherein said interferonincludes the mutations H57M, E58L, and Q61D.

Embodiment 25: The construct of embodiment 16, wherein said interferonincludes the mutations H57Y, E58L, and Q61D.

Embodiment 26: The construct of embodiment 16, wherein said interferonincludes the mutations H57Y, E58A, and Q61S.

Embodiment 27: The construct of embodiment 16, wherein said interferonincludes the mutations H57A, E58A, and Q61A.

Embodiment 28: The construct according to any one of embodiments 1-27,wherein said antibody includes the complementarity determining regionsof the B-B4 monoclonal antibody.

Embodiment 29: The construct of embodiment 28, wherein said antibodyincludes the VH and/or VL domain of the B-B4 monoclonal antibody.

Embodiment 30: The construct according to any one of embodiments 1-29,wherein said antibody is an antibody selected from the group consistingof is a single chain Fv (scFv), a FAB , a (Fab′)2, an (ScFv)₂, and afull IgG.

Embodiment 31: The construct according to any one of embodiments 1-29,wherein said antibody is an scFv.

Embodiment 32: The construct according to any one of embodiments 1-29,wherein said antibody is a full IgG.

Embodiment 33: The construct of embodiment 29, wherein said antibody isthe B-B4 monoclonal antibody.

Embodiment 34: The construct according to any of embodiments 1-33,wherein said antibody is chemically coupled to said interferon.

Embodiment 35: The construct according to any of embodiments 1-33,wherein said antibody is directly joined to said interferon (e.g., atthe C terminus of the heavy chain, at the C terminus of the light chain,at the N terminus of the heavy chain, or at the N-terminus of the lightchain of the antibody).

Embodiment 36: The construct according to any of embodiments 1-33,wherein said antibody is directly joined to said interferon with apeptide linker (e.g., at the C terminus of the heavy chain, at the Cterminus of the light chain, at the N terminus of the heavy chain, or atthe N-terminus of the light chain of the antibody).

Embodiment 37: The construct of embodiment 36, wherein said peptidelinker is proteolysis resistant.

Embodiment 38: The construct of according to any one of embodiments36-37, wherein said peptide linker is fewer than 15 amino acids inlength.

Embodiment 39: The construct of according to any one of embodiments36-38, wherein said peptide linker is not (Gly₄Ser)₃.

Embodiment 40: The construct of embodiment 35, wherein the amino acidsequence of said peptide linker is selected from the group consisting ofGGG, GGS, GGGGS (SEQ ID NO:7), SGGGGS (SEQ ID NO:8), GGGGSGGGGS (SEQ IDNO:9), A EAAAK A (SEQ ID NO:10), A EAAAK EAAAK A (SEQ ID NO:11), A EAAAKEAAAK EAAAK A (SEQ ID NO:12), A EAAAK EAAAK EAAAK EAAAK A (SEQ IDNO:13), A EAAAK EAAAK EAAAK EAAAK EAAAK A (SEQ ID NO:14), AEAAAKEAAAKAG(SEQ ID NO:15), AEAAAKEAAAKAGS (SEQ ID NO:16), GGGGG (SEQ ID NO:17),GGAGG (SEQ ID NO:18), GGGGGGGG (SEQ ID NO:19), GAGAGAGAGA (SEQ IDNO:20), RPLSYRPPFPFGFPSVRP (SEQ ID NO:21), YPRSIYIRRRHPSPSLTT (SEQ IDNO:22), TPSHLSHILPSFGLPTFN (SEQ ID NO:23), RPVSPFTFPRLSNSWLPA (SEQ IDNO:24), SPAAHFPRSIPRPGPIRT (SEQ ID NO:25), APGPSAPSHRSLPSRAFG (SEQ IDNO:26), PRNSIHFLHPLLVAPLGA (SEQ ID NO:27), MPSLSGVLQVRYLSPPDL (SEQ IDNO:28), SPQYPSPLTLTLPPHPSL (SEQ ID NO:29), NPSLNPPSYLHRAPSRIS (SEQ IDNO:30), LPWRTSLLPSLPLRRRP (SEQ ID NO:31), PPLFAKGPVGLLSRSFPP (SEQ IDNO:32), VPPAPVVSLRSAHARPPY (SEQ ID NO:33), LRPTPPRVRSYTCCPTP (SEQ IDNO:34), PNVAHVLPLLTVPWDNLR (SEQ ID NO:35), CNPLLPLCARSPAVRTFP (SEQ IDNO:36), LGTPTPTPTPTGEF (SEQ ID NO:37), EDFTRGKL (SEQ ID NO:38), L EAAAREAAAR EAAAR EAAAR (SEQ ID NO:39), L EAAAR EAAAR EAAAR (SEQ ID NO:40), LEAAAR EAAAR (SEQ ID NO:41), L EAAAR (SEQ ID NO:42), EAAAR EAAAR EAAAREAAAR (SEQ ID NO:43), EAAAR EAAAR EAAAR (SEQ ID NO:44), EAAAR EAAAR (SEQID NO:45), and EAAAR (SEQ ID NO:46).

Embodiment 41: The construct of embodiment 35, wherein the amino acidsequence of said peptide linker is selected from the group consisting ofGGGGS (SEQ ID NO:7), SGGGGS (SEQ ID NO:8), AEAAAKEAAAKAG (SEQ ID NO:15),and AEAAAKEAAAKAGS (SEQ ID NO:16).

Embodiment 42: The construct of embodiment 35, wherein the amino acidsequence of said peptide linker is SGGGGS (SEQ ID NO:8).

Embodiment 43: The construct of embodiment 1, wherein said constructincludes the YNS mutant interferon attached to the B-B4 monoclonalantibody by a linker including or consisting of the amino acid sequenceSGGGGS (SEQ ID NO:8).

Embodiment 44: The construct of embodiment 43, wherein said interferonis attached to the end of CH3 by said linker.

Embodiment 45: The according to any one of embodiments 1-44, whereinsaid construct or a component thereof is a recombinantly expressedfusion protein.

Embodiment 46: A pharmaceutical formulation including a constructaccording to any of embodiments 1-45 in a pharmaceutically acceptableexcipient.

Embodiment 47: The pharmaceutical formulation according to embodiment46, wherein said formulation is a unit dosage formulation.

Embodiment 48: The pharmaceutical formulation according to embodiment46, wherein said formulation is a formulated for parenteraladministration.

Embodiment 49: The pharmaceutical formulation according to embodiment46, wherein said formulation is a formulated for administration via aroute selected from the group consisting of oral administration,intravenous administration, intramuscular administration, direct tumoradministration, inhalation, rectal administration, vaginaladministration, transdermal administration, and subcutaneous depotadministration.

Embodiment 50: A method of inhibiting growth and/or proliferation of acell that expresses or overexpresses CD138, said method includingcontacting said cell with a construct according to any of embodiments1-45, or a formulation according to any one of embodiments 46-49 in anamount sufficient to inhibit growth or proliferation of said cell.

Embodiment 51: The method of embodiment 50, wherein said cell is acancer cell.

Embodiment 52: The method of embodiment 51, wherein said cancer cell isa metastatic cell.

Embodiment 53: The method of embodiment 51, wherein said cancer cell isin a solid tumor.

Embodiment 54: The method of embodiment 51, wherein said cancer cell iscell produced by a cancer selected from the group consisting of multiplemyeloma, ovarian carcinoma, cervical cancer, endometrial cancer, kidneycarcinoma, gall bladder carcinoma, transitional cell bladder carcinoma,gastric cancer, prostate adenocarcinoma, breast cancer, prostate cancer,lung cancer, colon carcinoma, Hodgkin's and non-Hodgkin's lymphoma,chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL),acute myeloblastic leukemia (AML), a solid tissue sarcoma, coloncarcinoma, non-small cell lung carcinoma, squamous cell lung carcinoma,colorectal carcinoma, hepato-carcinoma, pancreatic cancer, and head andneck carcinoma.

Embodiment 55: The method of embodiment 51, wherein said cancer cell isa cell of a multiple myeloma.

Embodiment 56: The method according to any one of embodiments 50-55,wherein said method includes inhibiting, delaying and/or preventing thegrowth of a tumor and/or spread of malignant tumor cells.

Embodiment 57: The method according to any one of embodiments 50-56,wherein said contacting includes systemically administering saidconstruct or formulation to a mammal.

Embodiment 58: The method according to any one of embodiments 50-56,wherein said contacting includes administering said construct orformulation directly into a tumor site.

Embodiment 59: The method according to any one of embodiments 50-56,wherein said contacting includes administering said construct orformulation via a route selected from the group consisting of oraladministration, intravenous administration, intramuscularadministration, direct tumor administration, inhalation, rectaladministration, vaginal administration, transdermal administration, andsubcutaneous depot administration.

Embodiment 60: The method according to any one of embodiments 50-56,wherein said contacting includes administering said construct orformulation intravenously.

Embodiment 61: The method according to any one of embodiments 50-60,wherein said cell is a cell in a human.

Embodiment 62: The method according to any one of embodiments 50-60,wherein said cell is a cell in a non-human mammal.

Embodiment 63: The method of embodiment 50, wherein said cancer cell isa cell produced by a multiple myeloma.

Embodiment 64: The method of embodiment 50, wherein said contactingincludes systemically administering said construct to a mammal.

Embodiment 65: The method of embodiment 50, wherein said contactingincludes administering said construct directly into a tumor site.

Embodiment 66: The method of embodiment 50, wherein said contactingincludes intravenous administration of said construct.

Embodiment 67: The method of embodiment 50, wherein said cancer cell isa cancer cell in a human.

Embodiment 68: The method of embodiment 50, wherein said cancer cell isa cancer cell in a non-human mammal.

Embodiment 69: The method according to any one of embodiments 51-68,wherein said method further includes administering to said subject oneor more cytotoxic agents and/or radiation in an amount effective toreduce tumor load, and/or to inhibit, delay, or prevent, the growthand/or spread of tumor cells including CD138 expressing cells.

Embodiment 70: The method of embodiment 69, wherein said method includesadministering to said subject bortezomib (VELCADE®).

Embodiment 71: The method of embodiment 69, wherein said method includesadministering to said subject lenalidomide.

Embodiment 72: A method for inhibiting, delaying and/or preventing thegrowth of a tumor and/or spread of malignant tumor cells in a subject inneed thereof, said method including: administering to said subject aconstruct according to any of embodiments 1-45, or a formulationaccording to any one of embodiments 46-49; and administering to saidsubject one or more cytotoxic agents and/or radiation in an amounteffective to reduce tumor load, and/or to inhibit, delay, or prevent thegrowth and/or spread of tumor cells including CD138 expressing cells.

Embodiment 73: The method of embodiment 72, wherein said method includesadministering to said subject bortezomib (VELCADE®).

Embodiment 74: The method of embodiment 72, wherein said method includesadministering to said subject lenalidomide.

Embodiment 75: A formulation including a construct according to any ofembodiments 1-45, and bortezomib and/or lenalidomide.

Embodiment 76: A kit including: a formulation including a constructaccording to any of embodiments 1-45; and bortezomib and/orlenalidomide.

Embodiment 77: A synergistic combination of a construct according to anyof embodiments 1-45, and bortezomib and/or lenalidomide.

Embodiment 78: A nucleic acid that encodes a fusion protein, said fusionprotein including an interferon attached to an anti-CD138 single-chainantibody or to a polypeptide including an anti-CD138 chain antibody.

Embodiment 79: The nucleic acid of embodiment 78, wherein saidinterferon is an interferon as found in a construct according to any ofembodiments 1-45.

Embodiment 80: The nucleic acid according to any one of embodiments78-79, wherein said antibody is an anti-CD138 antibody as found in aconstruct according to any of embodiments 1-45.

Embodiment 81: The nucleic acid according to any one of embodiments78-80, wherein said nucleic acid encodes a construct or a component of aconstruct according to any of embodiments 1-45.

Embodiment 82: A cell including a nucleic acid that expresses a fusionprotein, said cell including a nucleic acid according to any ofembodiments 78-81.

Definitions

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The term also includes variants on the traditional peptidelinkage joining the amino acids making up the polypeptide. Preferred“peptides”, “polypeptides”, and “proteins” are chains of amino acidswhose alpha carbons are linked through peptide bonds. The terminal aminoacid at one end of the chain (amino terminal) therefore has a free aminogroup, while the terminal amino acid at the other end of the chain(carboxy terminal) has a free carboxyl group. As used herein, the term“amino terminus” (abbreviated N-terminus) refers to the free α-aminogroup on an amino acid at the amino terminal of a peptide or to theα-amino group (imino group when participating in a peptide bond) of anamino acid at any other location within the peptide. Similarly, the term“carboxy terminus” refers to the free carboxyl group on the carboxyterminus of a peptide or the carboxyl group of an amino acid at anyother location within the peptide. Peptides also include essentially anypolyamino acid including, but not limited to peptide mimetics such asamino acids joined by an ether as opposed to an amide bond.

An “antibody”, as used herein, refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. In certain embodiments, theimmunoglobulin genes are human immunoglobulin genes. Recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as myriad immunoglobulinvariable region genes. Light chains are typically classified as eitherkappa or lambda. Heavy chains are typically classified as gamma, mu,alpha, delta, or epsilon, which in turn define the immunoglobulinclasses, IgG, IgM, IgA, IgD and IgE, respectively.

A typical (native) immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these regions of thelight and heavy chains respectively.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases orexpressed de novo. Thus, for example, pepsin digests an antibody belowthe disulfide linkages in the hinge region to produce F(ab)′₂, a dimerof Fab which itself is a light chain joined to V_(H)-C_(H)1 by adisulfide bond. The F(ab)′₂ may be reduced under mild conditions tobreak the disulfide linkage in the hinge region thereby converting the(Fab′)₂ dimer into an Fab′ monomer. The Fab′ monomer is essentially anFab with part of the hinge region (see, Fundamental Immunology, W. E.Paul, ed., Raven Press, N.Y. (1993), for a more detailed description ofother antibody fragments). While various antibody fragments are definedin terms of the digestion of an intact antibody, one of skill willappreciate that such Fab′ fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology. Thus, the termantibody, as used herein also includes antibody fragments eitherproduced by the modification of whole antibodies or synthesized de novousing recombinant DNA methodologies, including, but are not limited to,Fab′₂, IgG, IgM, IgA, IgE, scFv, dAb, nanobodies, unibodies, anddiabodies. In various embodiments preferred antibodies include, but arenot limited to Fab′₂, IgG, IgM, IgA, IgE, and single chain antibodies,more preferably single chain Fv (scFv) antibodies in which a variableheavy and a variable light chain are joined together (directly orthrough a peptide linker) to form a continuous polypeptide.

In certain embodiments antibodies and fragments used in the constructsdescribed herein can be bispecific. Bispecific antibodies or fragmentscan be of several configurations. For example, bispecific antibodies mayresemble single antibodies (or antibody fragments) but have twodifferent antigen binding sites (variable regions). In variousembodiments bispecific antibodies can be produced by chemical techniques(Kranz et al. (1981) Proc. Natl. Acad. Sci., USA, 78: 5807), by“polydoma” techniques (see, e.g., U.S. Pat. No. 4,474,893), or byrecombinant DNA techniques. In certain embodiments bispecific antibodiesof the present invention can have binding specificities for at least twodifferent epitopes at least one of which is a tumor associate antigen.In various embodiments the antibodies and fragments can also beheteroantibodies. Heteroantibodies are two or more antibodies, orantibody binding fragments (e.g., Fab) linked together, each antibody orfragment having a different specificity.

An “antigen-binding site” or “binding portion” refers to the part of animmunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains are referred to as “hypervariable regions” which are interposedbetween more conserved flanking stretches known as “framework regions”or “FRs”. Thus, the term “FR” refers to amino acid sequences that arenaturally found between and adjacent to hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen binding “surface”. This surface mediates recognition andbinding of the target antigen. The three hypervariable regions of eachof the heavy and light chains are referred to as “complementaritydetermining regions” or “CDRs” and are characterized, for example byKabat et al. Sequences of proteins of immunological interest, 4th ed.U.S. Dept. Health and Human Services, Public Health Services, Bethesda,Md. (1987).

The term “interferon” refers to a full-length interferon or to aninterferon fragment (truncated interferon) or interferon mutant, thatsubstantially retains the biological activity of the full lengthwild-type interferon (e.g., retains at least 50%, or preferably at least60%, or preferably at least 70%, or preferably at least 80%, preferablyat least 90%, more preferably at least 95%, 98%, or 99% of thefull-length interferon in its free form (e.g., when not a component of achimeric construct). Interferons include type I interferons (e.g.,interferon-alpha and interferon-beta) as well as type II inteferons(e.g., interferon-gamma). The interferon (e.g., IFN-α) can be fromessentially any mammalian species. In certain preferred embodiments, theinterferon is from a species selected from the group consisting ofhuman, equine, bovine, rodent, porcine, lagomorph, feline, canine,murine, caprine, ovine, a non-human primate, and the like. In variousembodiments the mutated interferon comprises one or more amino acidsubstitutions, insertions, and/or deletions.

A single chain Fv (“sFv” or “scFv”) polypeptide is a covalently linkedV_(H):V_(L) heterodimer which, in certain embodiments, may be expressedfrom a nucleic acid including V_(H)- and V_(L)-encoding sequences eitherjoined directly or joined by a peptide-encoding linker. Huston et al.(1998) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. A number of approachesfor converting the naturally aggregated, but chemically separated lightand heavy polypeptide chains from an antibody V region into an sFvmolecule that will fold into a three dimensional structure substantiallysimilar to the structure of an antigen-binding site are known (see,e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405, and 4,956,778).

“CD138” also known as syndecan-1 is a protein that in humans is encodedby the SDC1 gene. The protein encoded by this gene is a transmembrane(type I) heparan sulfate proteoglycan and is a member of the syndecanproteoglycan family. The syndecans mediate cell binding, cell signaling,and cytoskeletal organization and syndecan receptors are required forinternalization of the HIV-1 tat protein. The syndecan-1 proteinfunctions as an integral membrane protein and participates in cellproliferation, cell migration and cell-matrix interactions via itsreceptor for extracellular matrix proteins. Altered syndecan-1expression has been detected in several different tumor types. Whileseveral transcript variants may exist for this gene, the full-lengthnatures of only two have been described to date. These two represent themajor variants of this gene and encode the same protein. Syndecan-1(CD138) is a surface proteoglycan consisting of long unbranchedglycosaminoglycan (GAG) chains covalently attached to a proteinbackbone.

The phrase “inhibition of growth and/or proliferation” of a cancer cellrefers to decrease in the growth rate and/or proliferation rate of acancer cell. In certain embodiments this includes death of a cancer cell(e.g. via apoptosis). In certain embodiments this term also refers toinhibiting the growth and/or proliferation of a solid tumor and/orinducing tumor size reduction or elimination of the tumor.

The term “cancer marker” refers to biomolecules such as proteins,carbohydrates, glycoproteins, and the like that are exclusively orpreferentially or differentially expressed on a cancer cell and/or arefound in association with a cancer cell and thereby provide targetspreferential or specific to the cancer. In various embodiments thepreferential expression can be preferential expression as compared toany other cell in the organism, or preferential expression within aparticular area of the organism (e.g. within a particular organ ortissue).

The terms “subject,” “individual,” and “patient” may be usedinterchangeably and refer to a mammal, preferably a human or a non-humanprimate, but also domesticated mammals (e.g., canine or feline),laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig), andagricultural mammals (e.g., equine, bovine, porcine, ovine). In variousembodiments, the subject can be a human (e.g., adult male, adult female,adolescent male, adolescent female, male child, female child) under thecare of a physician or other health worker in a hospital, psychiatriccare facility, as an outpatient, or other clinical context. In certainembodiments, the subject may not be under the care or prescription of aphysician or other health worker.

The phrase “cause to be administered” refers to the actions taken by amedical professional (e.g., a physician), or a person controllingmedical care of a subject, that control and/or permit the administrationof the agent(s)/compound(s) at issue to the subject. Causing to beadministered can involve diagnosis and/or determination of anappropriate therapeutic or prophylactic regimen, and/or prescribingparticular agent(s)/compounds for a subject. Such prescribing caninclude, for example, drafting a prescription form, annotating a medicalrecord, and the like. Where administration is described herein, “causingto be administered” is also contemplated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate the production and activity ofanti-CD138-IFNα. FIG. 1A: Schematic diagram of anti-CD138-IFNα fusionprotein. The fusion protein contains murine variable region of antibodyB-B4, which is specific for CD138, and human IgG1κ constant regions.Human IFNα2 is fused to the C-terminus of the antibody via a flexibleSGGGGS (SEQ ID NO:8) linker. FIG. 1B: The effect of IFNα andanti-CD138-IFNα on Daudi cells. Cells were treated for 72 h with0.0032-50 pM IFNα or 0.032-500 pM anti-CD138-IFNα, and cell viabilitywas measured using the MTS assay. The experiment was performed once withdata averaged from triplicates.

FIG. 2 shows the cytoreductive effects of anti-CD138-IFNα against HMCLs.The indicated cell lines were incubated with varying concentrations (0.3pM-25 nM) of anti-CD20-IFNα or anti-CD138-IFNα for 3 days. Cellviability was measured using the MTS assay. The experiment was performedin triplicate for each concentration.

FIGS. 3A-3C show the effects of IFNα, anti-CD138-IFNα andanti-CD138-mutIFNα on six sensitive HMCLs. FIG. 3A: The effect oftreatments on cell viability. HMCLs were treated with 0.00002 pM-1 nM ofIFNα, anti-CD138-IFNα and anti-CD138-mutIFNα for 3 days (OCI-My 5, U266,H929 and MM144), 4 days (8226/S) or 7 days (ANBL-6) and analyzed forcell viability by MTS assay. The experiment was performed in triplicatefor each concentration. FIG. 3B: Cell cycle analysis followingtreatment. HMCLs were treated with 500 pM of the indicated proteins for4 days. Following permeabilization and staining with PI, DNA content wasanalyzed by flow cytometry. FIG. 3C: Determination of induction ofapoptosis following treatment. HMCLs were treated with 500 pM of theindicated proteins for 3 days and then stained with Alexa Fluor 488labeled Annexin V and PI and then analyzed by flow cytometry. Thenumbers in the upper right quadrant indicate the percentage of AnnexinV⁺PI⁺ apoptotic cells.

FIGS. 4A and 4B show the effect of IFNα, anti-CD138 IgG, anti-CD138-IFNαand anti-CD138-mutIFNα on proliferation and induction of senescence inOCI-My 5 cells. FIG. 4A: Cells were treated for 3 days with 0.3 pM-25 nMof the indicated proteins. The proliferative status of the cells wasdetermined by measuring ³[H]-thymidine incorporation. FIG. 4B: Cellswere treated with 1 nM of the indicated proteins for 3 days. Cleavage ofthe substrate C₁₂FDG was detected by flow cytometry as an indication ofβ-gal activity at pH 6. The percentage of cells with β-gal activity isshown. The experiment was also performed at 4, 6 and 7 days aftertreatment with similar results.

FIG. 5, panels A and B show activity of anti-CD138, anti-CD138-IFNαanti-CD138-mutIFNα against primary myeloma cells. (A) Purified MM cellsfrom 5 different patients were incubated with 25 nM of the indicatedproteins for 72 h and cell viability determined by trypan blue exclusionand an MTT assay. (B) Patient samples were treated with the indicatedproteins for 24, 48 or 72 h and the percentage of viable cells recovereddetermined by trypan blue exclusion and an MTT assay. Data represent theaverage of 6 patients. Both experiments were performed once.

FIGS. 6A and 6B, show activity of anti-CD138, anti-CD138-IFNαanti-CD138-mutIFNα in a xenograft model of MM. FIG. 6A: SCID mice wereinjected subcutaneously with 1×10⁷ OCI-My 5 cells and treated on days14, 16 and 18 as indicated by the black arrows with 100 μg of theindicated proteins. Survival and tumor growth were monitored. n=8. FIG.6B: NSG mice were injected subcutaneously with 1×10⁷ U226 cells andtreated on days 14, 16 and 18 as indicated by the black arrows with 100μg of the indicated proteins. One group was treated three additionaltimes with anti-CD138-mutIFNα on days 25, 32 and 63 as indicated by thegrey arrows. Survival and tumor growth were monitored. n=8.

FIG. 7 comparison of anti-proliferative activity of anti-CD20-IFNα withanti-CD20-mut-IFNα.

FIG. 8 comparison of anti-proliferative activity of anti-CD138-IFNα withanti-CD138-mut-IFNα.

FIG. 9 shows a comparison of the ability the anti-CD138-mutIFNα and theanti-CD138-IFNα in inhibiting IRF4 protein expression in U266 cells.U266 cells were treated with 1 nM of the indicated proteins for 2 daysand the expression level of IRF4 determined by Western blotting. Afternormalizing to GAPDH, the relative level of IRF4 expression wasquantified using densitometry.

FIG. 10 shows a comparison of the ability the anti-CD138-mutIFNα and theanti-CD138-IFNα in inhibiting its expression of cyclin D expression inU266 cells. U266 cells were treated with 1 nM of the indicated proteinsfor 2 days and the expression level of cyclin D determined by Westernblotting. After normalizing to GAPDH, the relative level of cyclin Dexpression was quantified using densitometry.

FIG. 11 shows a comparison of anti-CD138-mutIFNα2 and anti-CD138-IFNα2in inducing senescence in H929 and OCI-My5 myeloma cells. Senescentcells are metabolically active in vitro, but do not divide. One commonmarker for the detection of senescence is beta galactosidase (β-gal)activity at pH 6, which is a barometer for increased lysosomal contentof senescent cells. Cells were treated with 1 nM of the indicatedproteins for 3 days. Cleavage of the substrate C12FDG was detected byflow cytometry as an indication of β-gal activity at pH 6. Thepercentage of cells with β-gal activity is shown.

FIG. 12 shows a comparison of anti-CD138-mutIFNα2 and anti-CD138-IFNα2in inducing apoptosis in U266 and H929 cells. Cell lines were treatedwith 500 pM of the indicated proteins for 3 days and the percentage ofannexin V apoptotic cells was determined.

FIGS. 13A and 13B illustrate production of IFNα2 fusion proteins. FIG.13A shows a schematic diagram of anti-CD138 fusion proteins containingIFNα2 or IFNα2^(YNS). The fusion proteins contain V regions of murineantibody B-B4, which is specific for CD138, and human IgG1κ constantregions. Human IFNα2 or IFNα2^(YNS) is fused to the C-terminus of theantibody via a SerGly₄Ser peptide linker. FIG. 13B shows SDS PAGEanalysis of reduced and unreduced purified fusion proteins and controlIgG.

FIG. 14, panels A-C, illustrates growth inhibition of HMCLs by IFNα2fusion proteins. Panel A) The indicated cell lines were incubated withvarying concentrations (0.3 pM-25 nM for all cell lines except OCI-My5which was tested at 0.3 pM-1 nM) of anti-CD20-IFNα2 or anti-CD138-IFNα2for 3 days. Cellular metabolic activity was measured using the MTSassay. The experiment was performed in triplicate for eachconcentration. Panels B and C) HMCLs were treated with 0.00002 pM-1 nMof IFNα2, anti-CD138, anti-CD138-IFNα2, IFNβ, anti-CD20-IFNα2^(YNS) andanti-CD138-IFNα2^(YNS) for 3 days (U266, OCI-My5, NCI-H929, andMM1-144), or 7 days (ANBL-6) and analyzed for metabolic activity by MTSassay. The experiment was performed in triplicate for eachconcentration. For all experiments, error bars indicate the standarddeviation of the measurements. The experiment shown in panel A isdifferent from that shown in panels B and C. The date shown in Panel Awere shown previously in FIG. 2.

FIGS. 15A and 15B show that treatment with IFNα2 and fusion proteinsinduces apoptosis. FIG. 15A) HMCLs were treated with 500 pM of theindicated proteins for 3 days and then stained with Alexa Fluor 488labeled Annexin V and PI and analyzed by flow cytometry to assessinduction of apoptosis at high dose. The number in the upper rightquadrants indicates the percentage of Annexin V⁺/PI⁺ cells. The numberin the lower right quadrants for OCI-My 5 cells indicates the percentageof Annexin V⁺/PI⁻ cells. FIG. 15B) HMCLs (except OCI-My5, which wastreated with 5 pM) were treated with 1 pM of the indicated proteins toassess apoptosis at low dose. Cells were treated for 3 days and thenstained with Alexa Fluor 488 labeled Annexin V and PI and analyzed byflow cytometry as described above. Some of the data are shown above inFIG. 3C.

FIGS. 16A-16D show that IFNα2 and fusion proteins can induce alterationsin cell cycle progression and senescence in some HMCLs. FIG. 16A) HMCLswere treated with 500 pM of the indicated proteins for 4 days. Followingpermeabilization and staining with PI, DNA content was analyzed by flowcytometry. Shaded histograms are untreated cells, and solid lines aretreated cells. The percentages of cells in different phases of the cellcycle are shown in Table 5. Some of the data are shown in FIG. 3B. FIG.16B) Cells were treated with for 48 hours with 1 nM anti-CD138, IFNα2,anti-CD138-IFNα2, or anti-CD138-IFNα2^(YNS). Cell lysates were used todetermine the levels of ppRb and IRF-4 by Western blotting. Proteinloading was monitored by probing the same membranes with anti-GAPDH. Thebands from the blots were quantified using NIH Image J software, and theratio of GAPDH to either protein is shown. ND=not detected. U266 data of16B shown in FIG. 9. FIG. 16C) Cells were treated with 1 nM of theindicated proteins for 3 days. β-gal activity at pH 6, an indicator ofsenescence, was determined by following cleavage of the substrateC₁₂FDG. OCI-My5 data shown in FIG. 4B and again FIG. 11. H929 shown inFIG. 11. FIG. 16D) Cells were treated with 500 pM of the indicatedproteins for 3 days. Expression of Ki-67 was determined by using rabbitanti-Ki-67 followed by anti-rabbit IgG-FITC and analyzed by flowcytometry. Dashed lines are unstained cells. Shaded histograms areuntreated, stained cells and bold lines are treated, stained cells. Thepercentage of Ki-67 cells is shown.

FIGS. 17A and 17B show that fusion proteins confer protection to mice intwo xenograft models of multiple myeloma. FIG. 17A) Scid mice wereinjected subcutaneously with 1×10⁷ OCI-My5 cells and treated on days 14,16 and 18 as shown by the black arrows with 100 μg of the indicatedproteins. Survival and tumor growth were monitored. Eight mice weretreated for each group. One mouse survived to day 120. Similar data toFIG. 6A, but has p values. FIG. 17B) NSG mice were injectedsubcutaneously with 1×10⁷ U226 cells and treated on days 14, 16 and 18as indicated by the black arrows with 100 μg of the indicated proteins.Survival and tumor growth were monitored. Eight mice were treated foreach group. P values were calculated between groups. ⁻p≥0.05, *p<0.02,**p<0.008, ***p≤0.0007. Similar data in FIG. 6B but has p values anddoes not show all of the groups.

FIG. 18, panels A and B show that fusion proteins are effective againstprimary myeloma cells. Panel A) Purified multiple myeloma cells fromseven patients were incubated with 25 nM of the indicated proteins for72 hours. Cell viability was determined by trypan blue staining bycomparing the number of recovered viable cells to that of untreatedcontrol cells. Data also shown in FIG. 5A. Panel B) Purified multiplemyeloma cells from 7 patients were incubated with 100 nM of theindicated proteins for 72 hours. Cell viability was assessed asdescribed above.

FIG. 19 shows the results of an MTS assay following 3 day incubationwith the indicated proteins.

FIG. 20 shows the results of an MTS assay following 3 day incubationwith the indicated proteins.

FIG. 21 shows the results of an MTS assay following 3 day incubationwith the indicated proteins.

FIG. 22 shows the results of an MTS assay following 3 day incubationwith the indicated proteins.

FIG. 23 shows the results of an MTS assay following various combinationtreatments.

FIGS. 24-28 show flow cytometry results for apoptosis assays in variouscombination treatmetn using the IFN fusion proteins and VELCADE® asindicated.

FIG. 29 illustrates a synergy between anti-CD138-mutIFNα andlenalidomide.

DETAILED DESCRIPTION

Interferon alpha (IFNα) is an important cytokine in initiating theinnate immune response and also demonstrates a wide spectrum ofanti-tumor activities. The clinical use of interferon (e.g., IFNα) as ananticancer drug, however, is hampered by its short half-life, whichsignificantly compromises its therapeutic effect. In certain embodimentsthis invention pertains to the discovery that the therapeutic index ofinterferon can be improved by attaching the interferon to a targetingmoiety that specifically/preferentially binds a marker on or associatedwith the target cell (e.g., a tumor cell). This permits the delivery ofhigher doses of interferon to the target site with fewer systemiccomplications. In certain embodiments the construct shows lower sideeffects/toxicity than an untargeted interferon.

In particular, anti-CD138-IFNα2 antibody fusion proteins wereconstructed. CD138, also known as syndecan-1, is a heparan sulfateproteoglycan expressed by multiple myeloma (MM) cell lines, and inpatients, it is expressed on malignant plasma cells in peripheral bloodand in the bone marrow (Wijdenes 1996, Ridley 1993, Chilosi 1999). Theectodomain of CD138 is secreted into the serum in vivo (Dhodapkar 1997),and is shed by cultured cells (Subramanian 1997). CD138 mediatesadhesion to growth factors (Kiefer 1990) and extracellular matrixcomponents (Bernfield 1992) such as type I collagen (Ridley 1993).

As explained below and in the Example, targeting of human IFN (e.g.,IFNα) via the anti-CD138 antibody moiety improves the cytoreductiveeffects of the cytokine. Multiple mechanisms of action such as inductionof apoptosis, blockage in cell cycle progression and induction ofsenescence appear to be involved as HMCLs have different responses totreatment with fusion protein as explained below. It is alsodemonstrated that the fusion protein is effective against primary humanmyeloma patient cells and against MM tumors in a mouse xenograft model.In addition, mutations that increase the affinity of IFNα for IFNARresulted in a more effective molecule in vitro and in vivo.

Antibody fusions were produced by genetically fusing IFN to the end ofCH3 of an IgG1κ antibody molecule. A schematic of this fusion proteinspecific for CD138 is illustrated in FIG. 1A.

As illustrated in the Examples, the proteins were expressed followinggene transfection into Chinese hamster ovary (CHO) cells and werepurified using protein A sepharose or agarose. The proteins expressedfrom the transfected genes were of the appropriate size and areassembled into H2L2 molecules.

A mutant IFNα (mutated IFNα2 or IFNα2^(YNS) having the mutations H57Y,E58N, and Q61S (YNS) (see, e.g., Kalie et al. (2007) J. Biol. Chem.,282: 11602-11611)) was fused to the terminus of CH3 in an identicalfashion used to fuse wild-type IFNα. The mutant IFNα has higher affinityfor IFNAR.

A first construct was made comprising an antibody specific for CD20.Based on the published data for anti-CD20 antibodies we expected it toexhibit higher anti-proliferative activity against CD20 expressinglymphoma cells than fusion proteins containing wild-type IFNα. However,instead we found that it was less effective (see FIG. 7).

When we changed the binding specificity of the fusion proteinscontaining mutIFNα from CD20 to CD138 (using an anti-CD138 antibody), toour surprise we found that they were more effective than thosecontaining wild-type IFNα in inhibiting the proliferation of mostmyeloma cell lines. Shown in FIG. 8 are the results using myeloma cellline U266. Thus, anti-CD138-mutIFNα was more effective in inhibiting theproliferation of myeloma cells than anti-CD138-IFNα.

IRF4 is a protein shown to be essential for the survival of myelomacells. The anti-CD138-mutIFNα construct was shown to be more effectivethan the anti-CD138-IFNα construct in inhibiting its expression in U266cells (see, e.g., FIG. 9).

Increased expression of cyclin D has been shown to be associated withthe growth of myeloma cells and anti-CD138-mutIFNα was shown to be moreeffective than anti-CD138-IFNα in inhibiting its expression in U266cells (see FIG. 10).

Cellular senescence is one pathway for inhibiting tumor growth.Anti-CD138-mutIFNα was shown to be more effective than anti-CD138-IFNαin inducing cellular senescence in H929 and OCI-My5 myeloma cells (seeFIG. 11).

Apoptosis is one mechanism for inhibiting tumor growth. Theanti-CD138-mutIFNα construct was shown to be more effective than theanti-CD138-IFNα construct in inducing apoptosis in U266 and H929 myelomacells (see FIG. 12).

An important readout is which protein is most effective against primarymyeloma cells. Anti-CD138-mutIFNα was shown to be more effective thananti-CD138-IFNα against primary myeloma cells from patients (see FIG.5).

In vivo efficacy in a treatment model is very important.Anti-CD138-mutIFNα was shown to be more effective than anti-CD138-IFNαin treating tumors in mice bearing xenografts of either OCI-MY5 or U266cells (see FIG. 6).

In view of these findings it is believed that anti-CD138-interferonconstructs, and in particular anti-CD138-mutant interferon constructsshow surprising and unexpected efficacy against cells expressing or overexpressing CD138.

Thus, in certain embodiments, the constructs (e.g., chimeric moieties)comprising an interferon (e.g., IFN-α, IFNβ, mutant IFNα, mutant IFNβ,truncated IFNα, truncated IFNβ, etc.) attached to a targeting moiety(e.g., to an antibody that specifically binds CD138). The constructsinclude chemical conjugates as well as fusion proteins. Also providedare nucleic acids encoding the fusion proteins as well as cellstransfected with the nucleic acids to express the fusion proteins. Alsoprovided are methods of inhibiting growth and proliferation of cellsthat express or overexpress CD138 using the constructs described herein.In certain embodiments, the cells that express or over express CD138 arecancer cells. Accordingly in various embodiments, methods are providedfor inhibiting, delaying and/or preventing the growth of a tumor and/orspread of malignant tumor cell using the constructs described herein. Inaddition, kits comprising the constructs are provided, e.g., for thetreatment of various cancers.

I. Constructs comprising a targeting moiety attached to an interferon.

It was a surprising discovery that constructs (chimeric constructs)comprising a targeting moiety that binds (e.g., that preferentially orspecifically binds) to CD138 attached to a native (wildtype) or modifiedIFN (e.g., mutant IFN-α) can be effectively used to inhibit the growthand/or proliferation of target cells (e.g., cancer cells) that expressor overexpress CD138. In certain embodiments the CD138 targetingmoieties are chemically conjugated to the interferon, while in otherembodiments, the CD138 targeting moiety (or a component thereof) isexpressed as a fusion protein with the interferon. When produced as afusion protein the CD138 targeting moiety (e.g., antibody) (or acomponent thereof) can be directly fused to the interferon or attachedby means of a peptide linker (e.g., a (Gly₄Ser)₃ (SEQ ID NO:1) linker, aGly₄Ser (SEQ ID NO:2) linker, a SerGly₄Ser linker (SEQ ID NO:8), anAEAAAKEAAAKA (SEQ ID NO:15) linker, and the like.

A) CD138 Targeting moieties.

In various embodiments, the targeting moiety is a molecule thatspecifically or preferentially binds CD138 expressed, or overexpressed,by (e.g., on the surface of) or associated with the target cell(s)(e.g., cancer cells such as multiple myeloma cells). While essentiallyany cell that expresses or overexpresses CD138 can be targeted, certainpreferred cells include those associated with a pathology characterizedby hyperproliferation of a cell (i.e., a hyperproliferative disorder).

Hyperproliferative disorders characterized as cancer include but are notlimited to solid tumors, proliferation of metastatic cells, and thelike.

While the examples focus on the use of the constructs to inhibit/killmultiple myeloma cells, it has been recognized that CD138 isexpressed/overexpressed in a number of other cancers. Thus for example,CD138 has been shown to be expressed/overexpressed on the followingcancers: ovarian carcinoma, cervical cancer (Numa et al. (2002) Int. J.Oncol., 20(1): 39-43.), endometrial cancer (Choi et al. (2007) Int. J.Cancer, 121(4): 741-750), kidney carcinoma, gall bladder, transitionalcell bladder carcinoma, gastric cancer (Wiksten et al. (2008) Gastric:Anticancer Res. 28(4C): 2279-2287), prostate adenocarcinoma (Zellwegeret al. (2003) Prostate 55(1): 20-29), mammary carcinoma (Loussouarn etal. (2008) Br. J. Cancer, 28: 1993-1998), non-small cell lung carcinoma(Shah et al. (2004) Cancer 101(7): 1632-1638), squamous cell lungcarcinoma (Toyoshima et al. (2001) Lung Cancer, 31(2-3): 193-202), coloncarcinoma cells and cells of Hodgkin's and non-Hodgkin's lymphomas,colorectal carcinoma (Hashimoto et al. (2008) BMC Cancer 8: 185),hepato-carcinoma (Li et al. (2005) World J. Gastroenterol. 11(10):1445-1451), chronic lymphocytic leukemia (CLL), pancreatic (Conejo etal. (2000) Int. J. Cancer, 88(1): 12-20), and head and neck carcinoma(Anttonen et al. (1999) Br. J. Cancer, 79(3/4): 558-564) to name just afew. It is believed the methods described herein will be effective inthese various cancers and others.

These disorders have been well characterized in humans, but also existwith a similar etiology in other mammals, and can be treated byadministering the constructs described herein (alone or in the contextof a combined treatment plan (e.g., in combination with radiationtherapy and/or the use of other anti-cancer compounds).

Anti-CD138 Antibodies.

In certain embodiments, the targeting moieties can comprise antibodies,unibodies, or affybodies that specifically or preferentially bind CD138.Antibodies that specifically or preferentially bind CD138 are well knownto those of skill in the art and many are commercially available. Forexample Wijdenes et al. (1996) British Journal of Haematology, 94,318-323 describe an antibody that is specific for CD138 (syndecan-1) andthis antibody is commercially available from Abcam, Miltenyi Biotec, andthe like. Other illustrative and non-limiting anti-CD138 antibodiesinclude, but are not limited to the polyclonal rabbit anti-human CD138antibody LS-B3341 and the monoclonal mouse anti-Human CD138 AntibodyLS-B4051 available from LifeSpan Biosciences, Inc., monoclonal antibody(MI15) available from Pierce Antibodies, Biotest BT-062 anti-CD138, andthe like. Other anti-CD138 antibodies include, but are not limited toB-B2, 1D4, MI15 and 104-9 (see, e.g., Gattei et al. (1999) British J.Haematol., 104(1): 152-162).

In addition, anti-CD138 antibodies can be made using methods well knownto those of skill in the art. For example, antibodies can be produced byimmunizing an animal with CD138 or an immunogenic fragment thereof andraising the antibodies in that animal. Polyclonal antibodies can berecovered and used or converted to monoclonal antibodies according tomethods well known to those of skill in the art.

In certain embodiments, single chain anti-CD138 antibodies can becreated using a phage display library. One such method is described byFernandez et al. (2005) Journal of Clinical Oncology, 2005 ASCO AnnualMeeting Proceedings. 23(16S), Part I of II (June 1 Supplement), 2005:2550. The authors used combinatorial immunoglobulin (Ig) libraries withphage display to generate in vitro human Ig Fab fragments without theneed to maintain on-going hybridoma culture. A library of 10¹⁰ clonesfrom the cDNA of peripheral blood mononuclear cells of patients withadenocarcinoma were used to identify anti-CD138 specific Ig. Generallyfollowing removal of non-specific Fabs by exposing the Ig library to theepithelial cell line HEK, specific anti-CD138 Fabs were selected byexposing the Fab library to HEK transduced with human CD138. Six roundsof selection resulted in a panel of anti-CD138-bearing phage. Theanti-CD138-bearing phages bound multiple myeloma CD138+ cell lines(U266, SBN) by ELISA analysis while phage alone did not. Sequencing theFab VH and VL genes confirmed the heterogeneity of the panel ofanti-CD138 Fabs.

The constructs described herein need not be limited to the use of theantibodies described above, and other such antibodies as they are knownto those of skill in the art can be used in the constructs,formulations, and methods described herein.

In certain embodiments, the antibody has an affinity (K_(D)) for CD138of at least 1×10⁻⁶ M, or at least 1×10⁻⁷ M, or at least 1×10⁻⁸ M, or atleast 1×10⁻⁹ M, or at least 1×10⁻¹⁰ M, or at least 1×10⁻¹¹ M.

It will be recognized that the antibodies described above can beprovided as whole intact antibodies (e.g., IgG), antibody fragments, orsingle chain antibodies, using methods well known to those of skill inthe art. In addition, while the antibody can be from essentially anymammalian species, to reduce immunogenicity, it is desirable to use anantibody that is of the species in which the construct is to be used. Inother words, for use in a human, it is desirable to use a human,humanized, or chimeric human antibody.

While the above discussion pertains to antibodies, it will be recognizedthat affybodies and/or unibodies can be used instead of antibodies.

Unibodies.

UniBody are antibody technology that produces a stable, smaller antibodyformat with an anticipated longer therapeutic window than certain smallantibody formats. In certain embodiments unibodies are produced fromIgG4 antibodies by eliminating the hinge region of the antibody. Unlikethe full size IgG4 antibody, the half molecule fragment is very stableand is termed a uniBody. Halving the IgG4 molecule left only one area onthe UniBody that can bind to a target. Methods of producing unibodiesare described in detail in PCT Publication WO2007/059782, (see, also,Kolfschoten et al. (2007) Science 317: 1554-1557) and can be used tocreate unibodies based on any known anti-CD138 antibody.

Affibodies.

Affibody molecules are class of affinity proteins based on a 58-aminoacid residue protein domain, derived from one of the IgG-binding domainsof staphylococcal protein A. This three helix bundle domain has beenused as a scaffold for the construction of combinatorial phagemidlibraries, from which Affibody variants that target the desiredmolecules can be selected using phage display technology (see, e.g.,Nord et al. (1997) Nat. Biotechnol. 15: 772-777; Ronmark et al. (2002)Eur. J. Biochem., 269: 2647-2655.). Details of Affibodies and methods ofproduction are known to those of skill (see, e.g., U.S. Pat. No.5,831,).

B) Interferons

In various embodiments chimeric moieties of this invention comprise aninterferon (e.g., IFN-α, IFNβ, etc.) joined to the targeting moiety(e.g., anti-CD138 antibody). The interferon can be a full lengthwild-type interferon (e.g. IFN-α, IFNβ, IFN-γ, etc.) an interferonfragment (e.g., an IFN-α fragment), and/or a mutated interferon.Typically the interferon fragment is one that possesses the endogenousbinding affinity and/or activity of the native interferon, preferably ata level of at least 60%, or of at least 80%, more preferably at least90% or 95%, most preferably at least 98%, 99%, 100%, or a level greaterthan the wild-type interferon (in its isolated form).

Means of identifying such modified interferon molecules are routine tothose of skill in the art. In one illustrative approach, a library oftruncated and/or mutated IFN-α is produced and screened for IFN-αactivity. Methods of producing libraries of polypeptide variants arewell known to those of skill in the art. Thus, for example error-pronePCR can be used to create a library of mutant and/or truncated IFN-α(see, e.g., U.S. Pat. No. 6,365,408).

The resulting library members can then be screened according to standardmethods known to those of skill in the art. Thus, for example, IFN-αactivity can be assayed by measuring antiviral activity against aparticular test virus. Kits for assaying for IFN-α activity arecommercially available (see, e.g., ILITE™ alphabeta kit by Neutekbio,Ireland).

In various embodiments use of a mutated interferon alpha 2 (IFNα2) iscontemplated. Certain mutants include a mutation of the His at position57, and/or the E at position 58, and/or the Q at position 61. In certainembodiments the mutants include the mutation H57Y, and/or E58N, and/orQ61S. In certain embodiments the mutants include a mutated IFNα2 havingthe mutations H57Y, E58N, and Q61S (YNS) (see, e.g., Kalie et al. (2007)J. Biol. Chem., 282: 11602-11611).

In other embodiments mutants include a mutation of the His at position57, and/or the E at position 58, and/or the Q at position 61 to A(alanine). In certain embodiments the mutants include a mutated IFNα2having the mutations H57A, E58A, and Q61A (HEQ) (see, e.g., Jaitin etal. (2006) Mol. Cellular Biol., 26(5): 1888-1897). In certainembodiments the mutant interferon comprises a mutation of His atposition 57 to A, Y, or M, and/or a mutation of E at position 58 to A,or N, or D, or L, and/or a mutation of Q at position 61 to A, or S, orL, or D.

A mutated IFNβ comprising a serine substituted for the naturallyoccurring cysteine at amino acid 17 has also been demonstrated to showefficacy (see, e.g., Hawkins et al. (1985) Cancer Res., 45, 5914-5920.

In various embodiments use of truncated interferons is alsocontemplated. Human INFα, for example, with deletions of the first 15amino-terminal amino acid residues and/or the last 10-13carboxyl-terminal amino acid residues, have been shown to exhibitvirtually the same activity as the parent molecules (see, e.g., Ackerman(1984) Proc. Natl. Acad. Sci., USA, 81: 1045-1047). Accordingly the useof IFN-αs having 1, 2, 3, up to 13 carboxyl terminal amino acid residuesdeleted and/or 1, 2, 3, up to 15 amino terminal amino acid residuesdeleted are contemplated.

It has also been demonstrated that activity resides in huIFN-α fragmentHuIFN-α (1-110) (Id.). Accordingly carboxyl truncated IFNs withtruncations after residue 110 and/or with 1, 2, 3, up to 15 aminoterminal amino acid residues deleted are contemplated.

Certain C-terminally truncated interferon betas (IFNβ) have been shownto have increased activity (see, e.g., U.S. Patent Publication2009/0025106 A1). Accordingly, in certain embodiments the interferonused in the constructs described herein includes the C-terminallytruncated IFNβ described as IFN-Δ1, IFN-Δ2, IFN-Δ3, IFN-Δ4, IFN-Δ5,IFN-Δ6, IFN-Δ7, IFN-Δ8, IFN-Δ9, or IFN-Δ10 as described in U.S. PatentPublication NO: 2009/0025106 A1. In certain embodiments the interferonis IFN-Δ7, IFN-Δ8, or IFN-Δ9 (SEQ ID NOs: 57, 59, and 61 in US2009/0025106 A1 (see, Table 1).

TABLE 1 Truncated IFnβ showing enhanced activity (see U.S. PatentPublication 2009/0025106 A1). SEQ Truncated ID IFN Amino Acid SequenceNO IFN-Δ7 Met Gly Lys Met Ala Ser Leu Phe Ala Thr Phe Leu Val Val LeuVal 3 Ser Leu Ser Leu Ala Ser Glu Ser Ser Ala Cys Asp Leu Pro Gln ThrHis Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg IleSer Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu GluPhe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met IleGln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp GluThr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu GluAla Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu AspSer Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys GluLys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg SerPhe Ser Leu Ser Thr Asn Leu Gln IFN-Δ8 Met Gly Lys Met Ala Ser Leu PheAla Thr Phe Leu Val Val Leu Val 4 Ser Leu Ser Leu Ala Ser Glu Ser SerAla Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met LeuLeu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His AspPhe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr IlePro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys AspSer Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu TyrGln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr GluThr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln ArgIle Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val ValArg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu IFN-Δ9 Met GlyLys Met Ala Ser Leu Phe Ala Thr Phe Leu Val Val Leu Val 5 Ser Leu SerLeu Ala Ser Glu Ser Ser Ala Cys Asp Leu Pro Gln Thr His Ser Leu Gly SerArg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser CysLeu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln PheGln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe AsnLeu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp LysPhe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile GlnGly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala ValArg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser ProCys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser ThrAsn

In certain embodiments the use of chemically modified interferon is alsocontemplated. For example, in certain embodiments, the interferon ischemically modified to increase serum half-life. Thus, for example,(2-sulfo-9-fluorenylmethoxycarbonyl)₇-interferon-α2 undergoestime-dependent spontaneous hydrolysis, generating active interferon(see, e.g., Shechter et al. (2001) Proc. Natl. Acad. Sci., USA, 98(3):1212-1217). Other modifications, include for example, N-terminalmodifications including, but not limited to the addition of PEG,protecting groups, and the like. U.S. Pat. No. 5,824,784, for example,described N-terminally chemically modified interferon.

The foregoing interferons are intended to be illustrative and notlimiting. Using the teaching provided herein, other suitable modifiedinterferons (e.g., modified IFN-α, IFNβ, IFN-γ, etc.) can readily beidentified and produced.

C. Attachment of the targeting moiety (e.g., anti-CD138 antibody) to theInterferon.

In various embodiments, the targeting moiety (e.g., an anti-CD138antibody) and the interferon can be joined together in any order. Thus,for example, the antibody can be joined to either the amino or carboxyterminal of the interferon. The antibody can also be joined to aninternal region of the interferon, or conversely, the interferon can bejoined to an internal location or to any terminus of the antibody, aslong as the attachment does not interfere with binding of the antibodyto that target marker (e.g., CD138).

The antibody and the interferon (e.g., IFN-α, IFNβ, etc.) can beattached by any of a number of means well known to those of skill in theart. In certain embodiments, the interferon is conjugated, eitherdirectly or through a linker (spacer), to the antibody. In certainembodiments, however, it is preferable to recombinantly express theconstruct as a fusion protein (e.g., with a single chain antibody, orwith one chain of a multi-chain antibody).

i) Chemical conjugation of the targeting moiety to the interferon.

In certain embodiments, the targeting moiety (e.g., an anti-CD138antibody) is chemically conjugated to the interferon (e.g., IFN-α, IFNβ,mutIFNα, etc.) molecule. Means of chemically conjugating molecules arewell known to those of skill.

The procedure for conjugating two molecules varies according to thechemical structure of the agent. Polypeptides typically contain avariety of functional groups; e.g., carboxylic acid (COOH) or free amine(—NH₂) groups that are available for reaction with a suitable functionalgroup on the other peptide, or on a linker to join the moleculesthereto.

Alternatively, the antibody and/or the IFN-α can be derivatized toexpose or attach additional reactive functional groups. Thederivatization can involve attachment of any of a number of linkermolecules such as those available from Pierce Chemical Company, RockfordIll.

A “linker”, as used herein, typically refers to a molecule that is usedto join the antibody to the interferon. In various embodiments, thelinker is capable of forming covalent bonds to both the antibody and tothe interferon. Suitable linkers are well known to those of skill in theart and include, but are not limited to, straight or branched-chaincarbon linkers, heterocyclic carbon linkers, or peptide linkers. Incertain embodiments, the linker(s) can be joined to the constituentamino acids of the antibody and/or the interferon through their sidegroups (e.g., through a disulfide linkage to cysteine). In certainpreferred embodiments, the linkers are joined to the alpha carbon aminoand/or carboxyl groups of the terminal amino acids of the antibodyand/or the interferon.

A bifunctional linker having one functional group reactive with a groupon the antibody and another group reactive on the interferon, can beused to form the desired conjugate. Alternatively, derivatization caninvolve chemical treatment of the targeting moiety. Procedures forgeneration of, for example, free sulfhydryl groups on polypeptides, suchas antibodies or antibody fragments, are known (See U.S. Pat. No.:4,659,839).

Many procedures and linker molecules for attachment of various compoundsincluding radionuclide metal chelates, toxins and drugs to proteins suchas antibodies are known. See, for example, European Patent ApplicationNo. 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784;4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al. (1987)Cancer Res. 47: 4071-4075. In particular, production of variousimmunotoxins is well-known within the art and can be found, for examplein “Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet,”Thorpe et al., Monoclonal Antibodies in Clinical Medicine, AcademicPress, pp. 168-190 (1982); Waldmann (1991) Science, 252: 1657; U.S. Pat.Nos. 4,545,985 and 4,894,443, and the like.

ii) Production of fusion proteins.

In certain embodiments, a chimeric targeting moiety-interferon fusionprotein is synthesized using recombinant DNA methodology. Generally thisinvolves creating a DNA sequence that encodes the fusion protein,placing the DNA in an expression cassette under the control of aparticular promoter, expressing the protein in a host, isolating theexpressed protein and, if required, renaturing the protein.

DNA encoding the fusion proteins or encoding one chain of the antibodyattached to an interferon can be prepared by any suitable method,including, for example, cloning and restriction of appropriate sequencesor direct chemical synthesis by methods such as the phosphotriestermethod of Narang et al. (1979) Meth. Enzymol. 68: 90-99; thephosphodiester method of Brown et al. (1979) Meth. Enzymol. 68: 109-151;the diethylphosphoramidite method of Beaucage et al. (1981) Tetra.Lett., 22: 1859-1862); the solid support method of U.S. Pat. No.4,458,066, and the like.

Chemical synthesis produces a single stranded oligonucleotide. This canbe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Alternatively, subsequences can be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments can then be ligated to produce the desired DNA sequence.

In certain embodiments, DNA encoding fusion proteins can be cloned usingDNA amplification methods such as polymerase chain reaction (PCR). Thus,for example, the gene for IFN-α is PCR amplified, using a sense primercontaining the restriction site for, e.g., NdeI and an antisense primercontaining the restriction site for HindIII. This can produce a nucleicacid encoding the mature IFN-α sequence and having terminal restrictionsites. An antibody having “complementary” restriction sites cansimilarly be cloned and then ligated to the IFN-α and/or to a linkerattached to the IFN-α. Ligation of the nucleic acid sequences andinsertion into a vector produces a vector encoding IFN-α joined to theanti-CD138 antibody.

While the two molecules can be directly joined together, one of skillwill appreciate that the molecules can be separated by a peptide spacerconsisting of one or more amino acids. Generally the spacer will have nospecific biological activity other than to join the proteins or topreserve some minimum distance or other spatial relationship betweenthem. In certain embodiments, however, the constituent amino acids ofthe spacer can be selected to influence some property of the moleculesuch as the folding, net charge, or hydrophobicity.

It was a surprising discovery, however, that certain linkers areunsuitable for preparation of fusion proteins of the present invention.Thus, for example, the (Gly₄Ser)₃ (SEQ ID NO:1) linker was not wellsuited for the production of an anti-CD20-IFN-α construct. Without beingbound to a particular theory, it is believed the interferon was beingremoved from the fusion protein by proteolysis. Western blot analysisusing anti-Fc and anti-interferon, confirmed that both of the upperbands were heavy chains, but only the largest contained interferon.

Accordingly, in certain preferred embodiments, it is desirable to use alinker that is resistant to proteolysis. Certain preferred linkers arelinkers that are not the (Gly₄Ser)₃ (SEQ ID NO:6) linker. Certainpreferred linkers are linkers shorter than 15 amino acids, or linkersshorter than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acidsin length. In certain embodiments the linker is an alpha helical linkerranging in length up to about 12 or 13 or 14 amino acids in length.

Certain illustrative proteolysis -resistant linkers well suited for usein the constructs of this invention are shown in Table 2.

TABLE 2 Illustrative proteolysis-resistant linkers. Linker Seq SEQ ID NOGGG GGS GGGGS 7 SGGGGS 8 GGGGSGGGGS 9 A EAAAK A 10 A EAAAK EAAAK A 11 AEAAK EAAAK EAAAK A 12 A EAAAK EAAAK EAAAK EAAAK A 13 A EAAAK EAAAK EAAAKEAAAK EAAAK A 14 AEAAAKEAAAKAG 15 AEAAAKEAAAKAGS 16 GGGGG 17 GGAGG 18GGGGGGGG 19 GAGAGAGAGA 20 RPLSYRPPFPFGFPSVRP 21 YPRSIYIRRRHPSPSLTT 22TPSHLSHILPSFGLPTFN 23 RPVSPFTFPRLSNSWLPA 24 SPAAHFPRSIPRPGPIRT 25APGPSAPSHRSLPSRAFG 26 PRNSIHFLHPLLVAPLGA 27 MPSLSGVLQVRYLSPPDL 28SPQYPSPLTLTLPPHPSL 29 NPSLNPPSYLHRAPSRIS 30 LPWRTSLLPSLPLRRRP 31PPLFAKGPVGLLSRSFPP 32 VPPAPVVSLRSAHARPPY 33 LRPTPPRVRSYTCCPTP 34PNVAHVLPLLTVPWDNLR 35 CNPLLPLCARSPAVRTFP 36 LGTPTPTPTPTGEF 37 EDFTRGKL38 L EAAAR EAAAR EAAAR EAAAR 39 L EAAAR EAAAR EAAAR 40 L EAAAR EAAAR 41L EAAAR 42 EAAAR EAAAR EAAAR EAAAR 43 EAAAR EAAAR EAAAR 44 EAAAR EAAAR45 EAAAR 46

The nucleic acid sequences encoding the fusion proteins can be expressedin a variety of host cells, including E. coli, other bacterial hosts,yeast, and various higher eukaryotic cells such as the COS, CHO and HeLacells lines and myeloma cell lines. The recombinant protein gene istypically operably linked to appropriate expression control sequencesfor each host. For E. coli this includes a promoter such as the T7, trp,or lambda promoters, a ribosome binding site and preferably atranscription termination signal. For eukaryotic cells, the controlsequences will include a promoter and preferably an enhancer derivedfrom immunoglobulin genes, SV40, cytomegalovirus, etc., and apolyadenylation sequence, and may include splice donor and acceptorsequences.

The plasmids of the invention can be transferred into the chosen hostcell by well-known methods such as calcium chloride transformation forE. coli and calcium phosphate treatment or electroporation for mammaliancells. Cells transformed by the plasmids can be selected by resistanceto antibiotics conferred by genes contained on the plasmids, such as theamp, gpt, neo and hyg genes.

Once expressed, the recombinant fusion proteins can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes (1982) ProteinPurification, Springer-Verlag, N.Y.: Deutscher (1990) Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y., and the like). Substantially pure compositions of at leastabout 90 to 95% homogeneity are preferred, and 98 to 99% or morehomogeneity are most preferred for pharmaceutical uses. Once purified,partially or to homogeneity as desired, the polypeptides may then beused therapeutically.

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the fusion protein (e.g.,anti-CD138-IFN-α, anti-CD138-mutIFN-α, etc.) may possess a conformationsubstantially different than the native conformations of the constituentpolypeptides. In this case, it may be necessary to denature and reducethe polypeptide and then to cause the polypeptide to re-fold into thepreferred conformation. Methods of reducing and denaturing proteins andinducing re-folding are well known to those of skill in the art (see,e.g., Debinski et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitmanand Pastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al.(1992) Anal. Biochem., 205: 263-270). Debinski et al., for example,describe the denaturation and reduction of inclusion body proteins inguanidine-DTE. The protein is then refolded in a redox buffer containingoxidized glutathione and L-arginine.

In certain embodiments a transient expression system can be used toexpress the chimeric constructs described herein. Although many celllines potentially can be used, one cell line that works well fortransient expression is 293T. For transient expression of 293T on Day 0,9 million cells in 25 ml are seeded for each 150 mm tissue cultureplate. A 1 mg/ml of PEI (Polyethylenimine) is made using sterile water.For the expression of a complete antibody or antibody fusion protein, 25μg each of H and L (50 ug total) is used per plate. A volume of 5 ml isused for transfection of each 150 mm plate. The DNA is mixed with DMEM,the PEI is then added and the mixture is incubated at room temperaturefor 10 mins. 1.75 μg PEI is used for each ug of DNA. For transfection,the old medium is removed, discarded and replaced with 20 ml of freshmedium (Iscoves+5% calf serum). The transfection mix is added and theplate is swirled. On Day 2, the medium is replaced with 30 ml of Iscovesmedium containing 1% FBS (fetal bovine serum) to minimize the amount ofbovine Ig present. Supernatants are collected from the cells on Days 4,6and 13 by removing the medium and replacing it with 30 ml of freshIscoves containing 1% FBS.

The cloning and expression of an anti-CD138-IFN-α fusion protein isillustrated herein in Example 1.

One of skill would recognize these expression methods are illustrativeand not limiting. Modifications can be made to the fusion proteinsdescribed herein without diminishing their activity/efficacy. Somemodifications may be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids placed on either terminus tocreate conveniently located restriction sites or termination codons.

Other modifications can be made to increase serum half-life and/orbioavailability. Such modifications include, but are not limited to theincorporation of D amino acids (especially in the linker), the use ofnon-naturally occurring amino acids, pegylation of the fusion protein,and the like.

D. Other Multi-valent targeting moieties.

In certain embodiments this invention contemplates the use ofmultivalent, preferably trivalent, quadravalent, pentavalent or greatertargeting moieties to target the interferon to a target cell.

For example, multivalent anti-CD138 moieties can be produced by any of anumber of methods. For example, linkers having three, four, or morereactive sites can be reacted with anti-CD138 antibodies to form atrimer or greater conjugate.

In certain embodiments, phage display, yeast display, bacterial display,or other display systems can be used to express and display multiplecopies (e.g., at least 3, at least 4, at least 5, at least 6 copies,etc.) of a targeting antibody (e.g., anti-CD138 such as B-B4) andthereby effectively provide a multivalent targeting moiety.

In certain embodiments the use of diabodies and triabodies (e.g.,comprising two domains that bind CD-138 or one domain that binds CD138and another domain that binds, for example, a member of the EGFRreceptor family (e.g., EGFR, HER3, etc.). Typically, diabodies comprisea heavy (VH) chain variable domain connected to a light chain variabledomain (VL) on the same polypeptide chain (VH-VL) connected by a peptidelinker that is too short to allow pairing between the two domains on thesame chain. This forces pairing with the complementary domains ofanother chain and promotes the assembly of a dimeric molecule with twofunctional antigen binding sites (see, e.g., Holliger et al. (1993)Proc. Natl. Acad. Sci., 90: 6444-6448). In certain embodiments toconstruct bispecific diabodies the V-domains of antibody A and antibodyB are fused to create the two chains VHA-VLB, VHB-VLA. Each chain isinactive in binding to antigen, but recreates the functional antigenbinding sites of antibodies A and B on pairing with the other chain.

II. Combined uses.

The constructs described herein are useful for inhibiting the growthand/or proliferation of target cells (e.g., cancer cells). In variousembodiments the constructs can be used to inhibit disease progression,to reduce the rate of secondary tumor formation, to shrink tumor size,and/or to stabilize regression/remission.

Particularly in the treatment of cancer, the constructs, formulations,and methods described herein may also include additional therapeuticand/or pharmacologically acceptable agents. For instance, theconstructs, formulations, or methods may involve other agents for thetreatment of cancer. Such agents include, but are not limited toalkylating agents (e.g., mechlorethamine (Mustargen), cyclophosphamide(Cytoxan, Neosar), ifosfamide (Ifex), phenylalanine mustard; melphalen(Alkeran), chlorambucol (Leukeran), uracil mustard, estramustine(Emcyt), thiotepa (Thioplex), busulfan (Myerlan), lomustine (CeeNU),carmustine (BiCNU, BCNU), streptozocin (Zanosar), dacarbazine(DTIC-Dome), cis-platinum, cisplatin (PLATINOL® (cisplatin), PLATINOL®(cisplatin) AQ), carboplatin (Paraplatin), altretamine (Hexalen), etc.),antimetabolites (e.g. methotrexate (Amethopterin, Folex, Mexate,Rheumatrex), 5-fluoruracil (Adrucil, Efudex, Fluoroplex), floxuridine,5-fluorodeoxyuridine (FUDR), capecitabine (Xeloda), fludarabine:(Fludara), cytosine arabinoside (Cytaribine, Cytosar, ARA-C),6-mercaptopurine (Purinethol), 6-thioguanine (Thioguanine), gemcitabine(Gemzar), cladribine (Leustatin), deoxycoformycin; pentostatin (Nipent),etc.), antibiotics (e.g. doxorubicin (Adriamycin, Rubex, Doxil,Daunoxome-liposomal preparation), daunorubicin (Daunomycin, Cerubidine),idarubicin (Idamycin), valrubicin (Valstar), mitoxantrone (Novantrone),dactinomycin (Actinomycin D, Cosmegen), mithramycin, plicamycin(Mithracin), mitomycin C (Mutamycin), bleomycin (Blenoxane),procarbazine (Matulane), etc.), mitotic inhibitors (e.g. paclitaxel(Taxol), docetaxel (Taxotere), vinblatine sulfate (Velban, Velsar, VLB),vincristine sulfate (Oncovin, Vincasar PFS, Vincrex), vinorelbinesulfate (Navelbine), etc.), chromatin function inhibitors (e.g.,topotecan (Camptosar), irinotecan (Hycamtin), etoposide (VP-16, VePesid,Toposar), teniposide (VM-26, Vumon), etc.), hormones and hormoneinhibitors (e.g. diethylstilbesterol (Stilbesterol, Stilphostrol),estradiol, estrogen, esterified estrogens (Estratab, Menest),estramustine (Emcyt), tamoxifen (Nolvadex), toremifene (Fareston)anastrozole (Arimidex), letrozole (Femara), 17-OH-progesterone,medroxyprogesterone, megestrol acetate (Megace), goserelin (Zoladex),leuprolide (Leupron), testosteraone, methyltestosterone, fluoxmesterone(Android-F, Halotestin), flutamide (Eulexin), bicalutamide (Casodex),nilutamide (Nilandron), etc.), inhibitors of synthesis (e.g.,aminoglutethimide (Cytadren), ketoconazole (Nizoral), etc.),immunomodulators (e.g., RITUXIMAB® (Rituxan), trastuzumab (HERCEPTIN®),denileukin diftitox (Ontak), levamisole (Ergamisol), bacillusCalmette-Guerin, BCG (TheraCys, TICE BCG), interferon alpha-2a, alpha 2b(Roferon-A, Intron A), interleukin-2, aldesleukin (ProLeukin), etc.) andother agents such as 1-aspariginase (Elspar, Kidrolase), pegaspasgase(Oncaspar), hydroxyurea (Hydrea, Doxia), leucovorin (Wellcovorin),mitotane (Lysodren), porfimer (Photofrin), tretinoin (Veasnoid), and thelike.

III. Pharmaceutical Compositions.

In certain embodiments, in order to carry out the methods describedherein, one or more active agents (constructs described herein) areadministered, e.g. to an individual diagnosed as having (or at risk for)a cancer. The active agent(s) can be administered in the “native” formor, if desired, in the form of salts, esters, amides, prodrugs,derivatives, and the like, provided the salt, ester, amide, prodrug orderivative is suitable pharmacologically, i.e., effective in the presentmethod. Salts, esters, amides, prodrugs and other derivatives of theactive agents can be prepared using standard procedures known to thoseskilled in the art of synthetic organic chemistry and described, forexample, by March (1992) Advanced Organic Chemistry; Reactions,Mechanisms and Structure, 4th Ed. N.Y. Wiley-Interscience.

For example, acid addition salts are prepared from the free base usingconventional methodology that typically involves reaction with asuitable acid. Generally, the base form of the drug is dissolved in apolar organic solvent such as methanol or ethanol and the acid is addedthereto. The resulting salt either precipitates or can be brought out ofsolution by addition of a less polar solvent. Suitable acids forpreparing acid addition salts include both organic acids, e.g., aceticacid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malicacid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. An acid addition salt may be reconvertedto the free base by treatment with a suitable base. Particularlypreferred acid addition salts of the active agents herein are halidesalts, such as may be prepared using hydrochloric or hydrobromic acids.Conversely, preparation of basic salts of the active agents of thisinvention are prepared in a similar manner using a pharmaceuticallyacceptable base such as sodium hydroxide, potassium hydroxide, ammoniumhydroxide, calcium hydroxide, trimethylamine, or the like. Particularlypreferred basic salts include alkali metal salts, e.g., the sodium salt,and copper salts.

Preparation of esters typically involves functionalization of hydroxyland/or carboxyl groups which may be present within the molecularstructure of the drug. The esters are typically acyl-substitutedderivatives of free alcohol groups, i.e., moieties that are derived fromcarboxylic acids of the formula RCOOH where R is alky, and preferably islower alkyl. Esters can be reconverted to the free acids, if desired, byusing conventional hydrogenolysis or hydrolysis procedures.

Amides and prodrugs can also be prepared using techniques known to thoseskilled in the art or described in the pertinent literature. Forexample, amides may be prepared from esters, using suitable aminereactants, or they may be prepared from an anhydride or an acid chlorideby reaction with ammonia or a lower alkyl amine. Prodrugs are typicallyprepared by covalent attachment of a moiety that results in a compoundthat is therapeutically inactive until modified by an individual'smetabolic system.

The active agents (e.g., constructs) described herein are useful forparenteral, topical, oral, nasal (or otherwise inhaled), rectal, orlocal administration, such as by aerosol or transdermally, forprophylactic and/or therapeutic treatment of one or more of thepathologies/indications described herein (e.g., atherosclerosis and/orsymptoms thereof). The pharmaceutical compositions can be administeredin a variety of unit dosage forms depending upon the method ofadministration. Suitable unit dosage forms, include, but are not limitedto powders, tablets, pills, capsules, lozenges, suppositories, patches,nasal sprays, injectables, implantable sustained-release formulations,lipid complexes, etc.

In various embodiments the active agents (e.g., constructs) describedherein are typically combined with a pharmaceutically acceptable carrier(excipient) to form a pharmacological composition. Pharmaceuticallyacceptable carriers can contain one or more physiologically acceptablecompound(s) that act, for example, to stabilize the composition or toincrease or decrease the absorption of the active agent(s).Physiologically acceptable compounds can include, for example,carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, suchas ascorbic acid or glutathione, chelating agents, low molecular weightproteins, protection and uptake enhancers such as lipids, compositionsthat reduce the clearance or hydrolysis of the active agents, orexcipients or other stabilizers and/or buffers.

Other physiologically acceptable compounds include wetting agents,emulsifying agents, dispersing agents or preservatives that areparticularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, forexample, phenol and ascorbic acid. One skilled in the art wouldappreciate that the choice of pharmaceutically acceptable carrier(s),including a physiologically acceptable compound depends, for example, onthe route of administration of the active agent(s) and on the particularphysio-chemical characteristics of the active agent(s).

The excipients are preferably sterile and generally free of undesirablematter. These compositions may be sterilized by conventional, well-knownsterilization techniques.

In therapeutic applications, the constructs described herein orformulations comprising such constructs are administered to a subject,e.g., to patient suffering e.g. from a cancer, or at risk of cancer(e.g. after surgical removal of a primary tumor) in an amount sufficientto prevent and/or cure and/or or at least partially prevent or arrestthe disease and/or its complications. An amount adequate to accomplishthis is defined as a “therapeutically effective dose.” Amounts effectivefor this use will depend upon the severity of the disease and thegeneral state of the patient's health. Single or multipleadministrations of the compositions may be administered depending on thedosage and frequency as required and tolerated by the patient. In anyevent, the composition should provide a sufficient quantity of theactive agents of the formulations of this invention to effectively treat(ameliorate one or more symptoms) the patient.

The concentration of active agent(s) can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight andthe like in accordance with the particular mode of administrationselected and the patient's needs. Concentrations, however, willtypically be selected to provide dosages ranging from about 0.1 or 1mg/kg/day to about 50 mg/kg/day and sometimes higher. Typical dosagesrange from about 3 mg/kg/day to about 3.5 mg/kg/day, preferably fromabout 3.5 mg/kg/day to about 7.2 mg/kg/day, more preferably from about7.2 mg/kg/day to about 11.0 mg/kg/day, and most preferably from about11.0 mg/kg/day to about 15.0 mg/kg/day. In certain preferredembodiments, dosages range from about 10 mg/kg/day to about 50mg/kg/day. In certain embodiments, dosages range from about 20 mg toabout 50 mg given orally twice daily. It will be appreciated that suchdosages may be varied to optimize a therapeutic regimen in a particularsubject or group of subjects.

In certain embodiments, the active agents (e.g., constructs describedherein) are administered orally (e.g. via a tablet) or as an injectablein accordance with standard methods well known to those of skill in theart. In other preferred embodiments, the constructs may also bedelivered through the skin using conventional transdermal drug deliverysystems, i.e., transdermal “patches” wherein the active agent(s) aretypically contained within a laminated structure that serves as a drugdelivery device to be affixed to the skin. In such a structure, the drugcomposition is typically contained in a layer, or “reservoir,”underlying an upper backing layer. It will be appreciated that the term“reservoir” in this context refers to a quantity of “activeingredient(s)” that is ultimately available for delivery to the surfaceof the skin. Thus, for example, the “reservoir” may include the activeingredient(s) in an adhesive on a backing layer of the patch, or in anyof a variety of different matrix formulations known to those of skill inthe art. The patch may contain a single reservoir, or it may containmultiple reservoirs.

In one embodiment, the reservoir comprises a polymeric matrix of apharmaceutically acceptable contact adhesive material that serves toaffix the system to the skin during drug delivery. Examples of suitableskin contact adhesive materials include, but are not limited to,polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates,polyurethanes, and the like. Alternatively, the drug-containingreservoir and skin contact adhesive are present as separate and distinctlayers, with the adhesive underlying the reservoir which, in this case,may be either a polymeric matrix as described above, or it may be aliquid or hydrogel reservoir, or may take some other form. The backinglayer in these laminates, which serves as the upper surface of thedevice, preferably functions as a primary structural element of the“patch” and provides the device with much of its flexibility. Thematerial selected for the backing layer is preferably substantiallyimpermeable to the active agent(s) and any other materials that arepresent.

In certain embodiments elevated serum half-life can be maintained by theuse of sustained-release protein “packaging” systems. Such sustainedrelease systems are well known to those of skill in the art. In onepreferred embodiment, the ProLease™ biodegradable microsphere deliverysystem for proteins and peptides (see, e.g., Tracy (1998) Biotechnol.Prog. 14: 108; Johnson et al. (1996), Nature Med. 2: 795; Herbert et al.(1998), Pharmaceut. Res. 15, 357) a dry powder composed of biodegradablepolymeric microspheres containing the active agent in a polymer matrixthat can be compounded as a dry formulation with or without otheragents.

The ProLease™ microsphere fabrication process was specifically designedto achieve a high encapsulation efficiency while maintaining integrityof the active agent. The process consists of (i) preparation offreeze-dried drug particles from bulk by spray freeze-drying the drugsolution with stabilizing excipients, (ii) preparation of a drug-polymersuspension followed by sonication or homogenization to reduce the drugparticle size, (iii) production of frozen drug-polymer microspheres byatomization into liquid nitrogen, (iv) extraction of the polymer solventwith ethanol, and (v) filtration and vacuum drying to produce the finaldry-powder product. The resulting powder contains the solid form of theactive agents, which is homogeneously and rigidly dispersed withinporous polymer particles. The polymer most commonly used in the process,poly(lactide-co-glycolide) (PLG), is both biocompatible andbiodegradable.

Encapsulation can be achieved at low temperatures (e.g., −40° C.).During encapsulation, the protein is maintained in the solid state inthe absence of water, thus minimizing water-induced conformationalmobility of the protein, preventing protein degradation reactions thatinclude water as a reactant, and avoiding organic-aqueous interfaceswhere proteins may undergo denaturation. A preferred process usessolvents in which most proteins are insoluble, thus yielding highencapsulation efficiencies (e.g., greater than 95%).

In another embodiment, one or more components of the solution can beprovided as a “concentrate”, e.g., in a storage container (e.g., in apremeasured volume) ready for dilution, or in a soluble capsule readyfor addition to a volume of water.

The foregoing formulations and administration methods are intended to beillustrative and not limiting. It will be appreciated that, using theteaching provided herein, other suitable formulations and modes ofadministration can be readily devised.

IV. Kits.

In certain embodiments, kits for the treatment of a primary cancerand/or in an adjunct therapy are provided. In various embodiments thekits typically comprise a container containing a construct describedherein (e.g., anti-CD138-IFNα, anti-138-mutIFNα, anti-CD138-IFNβ, etc.).In various embodiments the construct can be present in apharmacologically acceptable excipient.

In addition the kits can optionally include instructional materialsdisclosing means of use of the chimeric moiety (e.g. to treat a cancerand/or as an adjunct therapeutic). The instructional materials may also,optionally, teach preferred dosages, counter-indications, and the like.

The kits can also include additional components to facilitate theparticular application for which the kit is designed. Thus, for example,in certain embodiments, the kit can additionally contain one or moreadditional anti-cancer drugs (e.g., doxirubicin, vinblastine, etc.), andthe like.

While the instructional materials typically comprise written or printedmaterials they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this invention. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Anti-CD138-IFNα and Anti-CD138-mutIFNα Constructs DemonstratePotent Apoptotic and Anti-Tumor Activities Against Multiple Myeloma

Materials and Methods

Cell Lines

HMCL cell lines were cultured in RPMI 1640 (Invitrogen, Carlsbad,Calif.) supplemented with 5% fetal calf serum (FCS; Atlanta Biologics,Lawrenceville, Ga.). ANBL-6 cells were cultured as described with theaddition of 2 ng/mL of IL-6. Chinese Hamster Ovary (CHO) cells werecultured in IMDM supplemented with 5% FCS. Daudi cells were purchasedfrom ATCC and grown in RPMI 1640 supplemented with 2 mM L-glutamine, 500μM β-mercaptoethanol, and 10% FCS.

Construction of Expression Vectors

The heavy and light chain variable region amino acid sequences of ananti-CD138 antibody were obtained from US Patent Publication No:2009/0175,863, which is incorporated herein by reference for theantibodies, and amino acid sequences (e.g., VH and VL sequences)described herein. A signal peptide with the amino acidsMGWSYIILFLVATATGVHS (SEQ ID NO:47) was added 5′ of the heavy chainvariable region. Similarly, a signal peptide with the amino acidsMKSQTQVFIFLLLCVSGAHG (SEQ ID NO:48) was added 5′ of the light chainvariable region. The amino acid sequences were sent to DNA 2.0 forcustom DNA synthesis using codons optimized for CHO expression. Thenucleotide sequence 5′ GGATATCCACC 3′ (SEQ ID NO:49), containing a Kozakribosomal recognition site, was also added 5′ of each sequence tofacilitate downstream cloning. To further facilitate downstream cloning,the sequence 5′ GCTAGCC 3′ (SEQ ID NO:50) was added 3′ of the heavychain variable region, and the sequence 5′ CGTAAGTCGACG 5′ (SEQ IDNO:51) was added 3′ of the light chain variable region.

The heavy chain variable region flanked by EcoRV and NheI restrictionsites was sequence verified and cloned into an expression vectorcontaining IgG1 alone, or IgG1 fused to human interferon alpha (hIFNα).The light chain variable region flanked by EcoRV and SalI restrictionsites was also sequence verified before cloning into an expressionvector containing a human kappa constant region.

VH sequence: (SEQ ID NO: 52) M G W S Y I I L F L V A T A T G V H S Q V QL Q Q S G S E L M M P G A S V K I S C K A T G Y T F S N Y W I E W V K QR P G H G L E W I G E I L P G T G R T I Y N E K F K G K A T F T A D I SS N T V Q M Q L S S L T S E D S A V Y Y C A R R D Y Y G N F Y Y A M D YW G Q G T S V T V S S VL sequence: (SEQ ID NO: 53) M K S Q T Q V F I F LL L C V S G A H G DI Q M T Q S T S S L S A S L G D R V T I S C S A S Q GI N N Y L N W Y Q Q K P D G T V E L L I Y Y T S T L Q S G V P S R F S GS G S G T D Y S L T I S N L E P E D I G T Y Y C Q Q Y S K L P R T F G GG T K L E I K

To construct anti-CD138-mutIFNα, nested polymerase chain reaction (PCR)was used to introduce three amino acid mutations—H57Y, E58N, and Q61S.The first round of PCR was done using the forward primer 5′-CGC GGA TCCTGT GAT CTG CCT CAA ACC CAC-3′ (SEQ ID NO:54) and reverse primer 5′-CCTCTA GAA TCA TTC CTT ACT TCT TAA ACT-3′ (SEQ ID NO:55). The nested PCRwas done using forward primer 5′-CCTGTCCTCTACAATATGATCTCACAGATCTTC-3′(SEQ ID NO:56) and reverse primer5′-GAAGATCTGTGAGATCATATTGTAGAGGACAGG-3′ (SEQ ID NO:57), which containthe mutations to IFNα. The insert was cloned into pCR2.1-TOPO vector(Invitrogen) and DNA sequence was verified. The XbaI/BamHI fragmentcontaining the mutIFNα sequence was cloned into an anti-CD20 human γl Hchain-IFNβ fusion vector pAH6747, yielding pAH11015. The BamHI/AvrIIfragment from pAH11015 was then used to replace the wild-type IFNαsequence from expression vector pAH6905, which contains the anti-CD138V_(H) yielding vector pAH11016.

Protein Production and Purification

Fusion proteins were produced in CHO cells by transfection of H and Lchain expression vectors. Stably transfected cells were isolated byselection on histidinol. For IgG and IgG fusion protein production,cells were seeded into roller bottles. At confluency, cells wereexpanded to 100 mL with IMDM+1% Fetal Clone (Thermo Fisher, Waltham,Mass.). The supernatant was removed every 2-3 days and replaced withfresh medium. Cell free culture supernatants were then passed through aprotein A-Sepharose 4B fast flow column (Sigma-Aldrich, St. Louis, Mo.)and the bound protein eluted with 0.1 M citric acid, pH 3.5. Elutedfractions were neutralized immediately with 2 M Tris-HCl pH 8.0.Fractions were run on SDS-PAGE gels and stained with Coomassie blue toverify protein purity and integrity. Concentrations of proteins weredetermined using the BCA assay (Pierce, Rockford, Ill.). Anti-CD20-IFNαused as untargeted control protein was produced as described previously(Xuan et al. (2010) Blood, 115: 2864-2871).

Cell Viability Assay

HMCLs were seeded in 96-well plates and incubated with 0.00002 pM-25 nMof anti-CD20-IFNα, IFNα, anti-CD138-IFNα or anti-CD138-mutIFNα oranti-CD138-IFNα at 37° C. in a 5% CO₂ atmosphere for 3, 4 or 7 days.Percent cell viability was determined using MTS solution (Promega,Madison, WI) by measuring absorbance at 490 nm using a Synergy HTMulti-Detection Microplate Reader (BioTek Instruments, Inc., Winooski,Vt.) with untreated cells being 100%. GraphPad Prism (GraphPad SoftwareInc., La Jolla, Calif.) was used to analyze data by non-linearregression with the log (inhibitor) vs. the response with a variableslope with the IC₅₀ calculated. Data are expressed as a percentage ofmaximum metabolic activity. The experiments were performed intriplicate.

Apoptosis Assay

Cells were incubated with 500 pM of IFNα, anti-CD138-IFNα oranti-CD138-mutIFNα for 3 days and stained with Alexa Fluor 488 labeledAnnexin V and propidium iodide (PI) using the Vybrant Apoptosis Kit #2(Molecular Probes, Carlsbad, Calif.) as per manufacturer's instructions.Cells were analyzed by flow cytometry using FlowJo software.

Cell Cycle Analysis

Cells were treated with 500 pM of IFNα, anti-CD138-IFNα, oranti-CD138-mutIFNα for 4 days. Cells were incubated with 1 ml ofhypotonic DNA staining buffer (1 mg/ml sodium citrate, 100 μg/ml PI, 20μg/ml RNase A, and 0.3% Triton X-100 in dH₂O) for 30-60 min at 4° C. inthe dark. Cells were analyzed by flow cytometry and cell cycle analysisperformed using FlowJo software with the Watson Model.

Cell Proliferation Assay

Cells were treated for 3 days with 0.3 pM-25 nM IFNα, anti-CD138-IFNα,or anti-CD138-mutIFNα. The proliferative status of the cells wasdetermined by measuring ³[H]-thymidine incorporation.

β-gal Activity as a Marker for Senescent Cells

Senescence induced β-galactosidase (β-gal) activity was detected asdescribed previously (Debacq-Chainiaux et al. (2009) Nature Protocols,4: 1798-1806). Briefly, cells were treated with 1 nM of anti-CD138 IgG,anti-CD138-IFNα or anti-CD138-mutIFNα for 3, 4, 6 or 7 days. To inducelysosomal alkalinization, cells were incubated in 100 nM bafilomycin A1(Sigma) for 1 h. Then cells were incubated for 30 min with the β-galsubstrate dodecanoylaminofluorescein di-β-D-galactopyranoside (C₁₂FDG;Invitrogen), which becomes fluorescent after cleavage by β-gal. Afterwashing twice with cold PBS, cells were resuspended in cold PBScontaining 1 mM phenylethyl thiogalactoside (PETG), a β-gal inhibitor,and analyzed by flow cytometry for C₁₂-fluorescein.

Treatment of Primary MM Cells from Patients

Patients with active myeloma were biopsied while off therapy and myelomacells isolated by negative antibody selection to >95% purity. Cells wereincubated with 25 or 100 nM of anti-CD138 IgG, anti-CD138-IFNα oranti-CD138-mutIFNα for 24, 48 or 72 h. Trypan blue and MTT assays wereused to determine the number of viable cells. The percentage of viablerecovered cells was calculated based upon the number of viable cellsrecovered from DMSO-treated control cells, which was arbitrarilydesignated as 100%.

Western Blots

Cells were treated for 48 h with anti-CD138, IFNα, IFNβ,anti-CD138-IFNα, or anti-CD138-mutIFNα. Cells were lysed using RIPAbuffer (50 mM Tris HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodiumdeoxycholate, 0.1% SDS) containing a protease inhibitor cocktail (RocheApplied Science, Indianapolis, Ind.). The cytosolic fractions werereduced with β-mercaptoethanol and separated by SDS-PAGE. Followingtransfer to nitrocellulose membrane (Whatman, Piscataway, N.J.) andblocking, samples were incubated with the following primary rabbitantibodies: anti-IRF4 (Epitomics, Berlingham, Calif.),anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma), anti-ppRbSer807/811 (Santa Cruz Biotechnology, Santa Cruz, Calif.), anti-cyclin D(Millipore, Billerica, Mass.), or anti-β tubulin (Abcam, Cambridge,Mass.). Secondary anti-rabbit IgG-HRP (GE Healthcare, Billerica, Mass.)was used and the blots were developed using enhanced chemiluminescence(ECL; Thermo Scientific, Waltham, Mass.).

In Vivo Anti-Tumor Activity Against OCI-My 5 and U266 Cells

Six- to eight-week old female SCID white mice were used to establishOCI-My 5 tumors. After washing in cold HBSS (Invitrogen), mice wereinoculated subcutaneously with 1×10⁷ cells in 200 μl of HBSS at the baseof the tail. Mice were treated intravenously with PBS, 100 μg ofanti-CD138, anti-dansyl (DNS)-IFNα, anti-CD138-IFNα, anti-CD20-mutIFNα,or anti-CD138-mutIFNα on days 14, 16, 18 post tumor challenge. Eightmice were included in each group. Bidirectional tumor growthmeasurements were obtained throughout the experiment, and mice weresacrificed when tumors reached 1.5 cm in diameter as per institutionalguidelines. All studies were performed in compliance with the U.S.Department of Health and Human Services Guide for the Care and Use ofLaboratory Animals and were approved by the UCLA Animal ResearchCommittee.

For U266 tumors, NOD scid IL-2 receptor gamma chain knock out (NSG) micewere injected subcutaneously with 1×10⁷ U226 cells and treated on days14, 16 and 18 with 100 μg of the indicated proteins. One group wastreated two additional times with anti-CD138-mutIFNα on days 25, 32 and63. Eight mice were treated for each group except 6 mice for the groupthat received six treatments with anti-CD138-mutIFNα. Survival wasmonitored as well as the size of the tumors.

Results

Production and Characterization of Anti-CD138-IFNα Fusion Proteins

The V_(H) and V_(L) of the anti-CD138 antibody B-B4 were cloned intovectors for the expression of human anti-CD138 IgG1 either unfused orfused to IFNα (FIG. 1A). The vectors were expressed in stabletransfectants of CHO cells, and the purified Igs were characterized withrespect to their size, and assembly status, and ability to bind antigen(data not shown). To test for IFNα activity, the proteins were testedagainst the Daudi human lymphoma cell line, which is highly sensitive tothe effects of IFNα. Cells were treated for 72 h and cell viability wasmeasured using the MTS assay. The anti-CD138-IFNα was just as effectiveas recombinant IFNα in its cytotoxic effects on Daudi cells (FIG. 1B),confirming that IFN activity was not affected by IFNα's fusion to IgG.

Anti-CD138-IFNα Fusion Protein is Effective Against Some MM Cell Lines

MM cell lines have been shown to differ in their responses to variousdrugs and treatment. They also show biological and genetic heterogeneityand have been classified using various methods (Carrasco et al. (2006)Cancer Cell 9: 313-325; Drexler et al. (2000) Leukemia, 14: 777-782;Lombardi et al. (2006) Genes Chromosomes Cancer, 46: 226-238; Moreaux etal. (2011) Haematologica, 96: 574-582). Therefore, we assembled a panelof thirteen MM cell lines (XG-1, XG-2, OPM-1, OPM-2, S6B45, Delta 47,8266/Dox40, 8266/S, H929, ANBL-6, MM144, U266, and OCI-My 5) toinvestigate their response to IFNα and the fusion protein. The panelincludes both hyperdiploid and nonhyperdiploid cells with variouschromosomal translocations (Table 3). Anti-CD138 IgG was found to bindall of the MM cell lines but did not bind to Daudi cells (data notshown), indicating that they express CD138 antigen.

TABLE 3 Panel of MM cell lines. Mode of Action of anti- CD138- Cell IFNαLine Ras TP53 RB MYC fusion XG-1 Hypodiploid 11q13::14q32 CCND1 mutabnormal 8q24::14q32 Monosomy 13 XG-2 Hyperdiploid 12q24.31:: Unknownmut abnormal 14q32 target (unknown MAFB target) insCλ@20q11 on 20t(11;14:?) (q13; q32; ?) OPM-1 Hyperdiploid der(4), CH insertion 74 der(14)of ins(Cq; 14) t(1; 8)(q12?; 1 i(ins(Cq; 14)) 24):der(8) OPM-2Hyperdiploid 4; 14 FGFR3 wt abnormal 8; 14 67 der(4), der and CHinsertion (14) MMSET on ins(Cq; 14) t(1; 8)(q12?; 1 wr):der(8) S6B45 Wtand mutated Delta 47 Hyperdiploid 11?p insCλ@8q24 45 on der(19)t(8; 19)(q13; q13) 8226/D ox 40 8226/S Hyperdiploid 14; 16 c-maf mut abnormalone c-myc Apoptosis Aka 60 t(1; 14)(p13; allelic insertion onRPMI8266*** q32) loss t(16; 22)(q23; 111):der(16) H929 Hyperdiploid14p16::14q32 Overexp mut Wt one 8q24::20q11 Apoptosis 45 FGFR3 allelicT(8; 20)q24; ?): Block in and loss der(8) cell cycle MMSET ANBL-6Hyperdiploid 14q32::16q23 Overexp Wt abnormal ? Apoptosis 82 c-maf MM14414; 16 Overexp ? ? ? Apoptosis c-maf Block in cell cycle U266*Hyperdiploid Insertion at* Overexp Wt abnormal biallelic LMYC (notApoptosis 39 Cyclin loss c-myc) D1 tp13::1p34 OCI- Hyperdiploid14q32::16q23 Overexp ? ? ? 8q24::14q32 & Block in My 5 46 c-mafdup(14)(q32q?22) cell cycle senescence† †Apoptosis and senescence wasobserved only with anti-CD138-mutIFNα treatment and not with IFNα aloneor anti-CD138-IFNα treatment *U266-Lombardi and Moreaux says t(11; 14)while Gabrea says 11q13 on der(11)t(11; ?)(q14; ?) ***This cell line maybe the same as RPMI-8226. There is conflicting data as Lombardi saysthat RPMI-8226 has complex rearrangement involving “c-MYC insertion ont(16; 22)(q32; q11):der(16)” but Moreaux says 14; 16 $Zhang (1994) Thedesignation t(A; B)(p1; q2) is used to denote a translocation betweenchromosome A and chromosome B. The information in the second set ofparentheses, when given, gives the precise location within thechromosome for chromosomes A and B respectively - with p indicating theshort arm of the chromosome, q indicating the long arm, and the numbersafter p or q refer to regions, bands and subbands seen when staining thechromosome with a staining dye. See also the definition of a “geneticlocus”.

To determine if the HMCLs were sensitive to treatment with IFNα fusionprotein, HMCLs were incubated with varying concentrations ofanti-CD138-IFNα, and cell viability was assessed. Anti-CD138-IFNα wasused to target IFNα to the MM cell lines while anti-CD20-IFNα was usedas an untargeted control. After 3 days of treatment, cell viability wasmeasured by MTS activity. The MM cell lines differed in their responseto treatment with the fusion proteins. The targeted anti-CD138-IFNα wasfound to be more effective than untargeted anti-CD20-IFNα for H929,ANBL-6, U266, MM144 and OCI-My 5 cell lines. The fusion protein hadlittle or no effect on the XG-1, XG-2, OPM-1, OPM-2, Delta 47 cell lineswhile an intermediate effect was observed for S6B45, 8266/Dox40, and8266S (FIG. 2). However, when treatment with anti-CD138-IFNα wasincreased to 4 days, a decrease in viability was observed in OPM-1,OPM-2 and Delta 47 cells (data not shown). Similar results have beenpublished for OPM-1, OPM-2, XG-1, Delta 47, ANBL-6, MM144, and H929 whenthese cell lines were treated with IFNα, but in contrast to our data,XG-2 was found to be responsive to IFNα (Crowder et al. (2005) Blood,105: 1280-1287). Our data demonstrate that addition of anti-CD138 IgG toIFNα results in a fusion protein effective against a number of HMCLsrepresenting MM with different molecular abnormalities.

The Effects of IFNα, Anti-CD138-IFNα and Anti-CD138-mutIFNα FusionProteins on HMCLs

IFNα and IFNβ share a common receptor comprised of two transmembraneproteins, IFNAR1 and IFNAR2, and have many overlapping activities.However, differential activities of the various type I IFNs have alsobeen reported. IFNβ has been reported to have a ˜20- to 50-fold greateraffinity for IFNAR1 than IFNα2 and this greater affinity has been shownto correlate with a significantly higher anti-proliferative activity(Jaitin et al. (2006) Mol Cell Biol 26: 1888-1897; Kalie et al. (2007)J. Biol. Chem., 282: 11602-11611). In an initial attempt to determine ifIFNβ fusion proteins would be an effective anti-cancer therapeutic, wefused mouse or human IFNβ to the C-terminal end of an anti-tumor IgG.Although the mouse IFNβ fusion protein retained its activity, the humanIFNβ fusion protein had a >100-fold decrease in activity (unpublishedresults). As an alternative approach, we chose to make an anti-CD138fusion protein using a mutant IFNα2 designed to mimic IFNβ with greateraffinity for IFNAR1 and enhanced anti-proliferative activity (Kalie etal. (2007) J. Biol. Chem., 282: 11602-11611). This mutant IFNα2(mutIFNα) contains mutations at three positions (H57Y, E58N, Q61S),which confer upon it a 60-fold increased affinity for IFNAR1 thanwild-type IFNα2 and a 3-fold higher affinity than IFNβ. In addition,mutIFNα was shown to exhibit an increase in anti-proliferative activity(80- and 150-fold increase, depending on the cell line used) whencompared to wild-type IFNα, and a slight increase in activity whencompared to IFNβ. Surprisingly, targeted anti-CD20-mutIFNα was found tobe less effective in inhibiting the proliferation of CD20 expressingDaudi cells than targeted wild type IFNα (anti-Cd20-IFNα). Theanti-viral activity of wild-type IFNα, mutIFNα and IFNβ were found to bevery similar (Id.).

The anti-tumor activities of IFNα, anti-CD138-IFNα andanti-CD138-mutIFNα against six sensitive cell lines were examined. Morespecifically, we examined their ability to inhibit cell proliferation,influence cell cycle progression, and cause apoptosis. The cytoreductiveactivity of anti-CD138-IFNα and anti-CD138-mutIFNα was compared tountargeted IFNα by MTS assay. The targeted fusion proteins were moreeffective at decreasing cell viability than IFNα alone for all HMCLsexcept H929, in which IFNα and the fusion proteins had very similaractivity. When compared to one another, anti-CD138-IFNα andanti-CD138-mutIFNα showed similar effects in 8226/S, MM144 and H929.Anti-CD138-IFNα was more effective than anti-CD138-mutIFNα for theANBL-6 cell line. In contrast, anti-CD138-mutIFNα was more effectivethan anti-CD138-IFNα in U266 and OCI-My5 cells (FIG. 3A).

The cytoreductive effects of the IFNα fusion proteins were furtherinvestigated by determining if HMCLs were blocked in cell cycleprogression or were undergoing apoptosis. To determine if there werechanges to the cell cycle, HMCLs were analyzed for DNA content by flowcytometry following permeabilization and staining with PI after 4 daysof treatment (FIG. 3B). 8226/S, ANBL-6 and U266 cells did not exhibitany changes in cell cycle or DNA content in response to IFNα or fusionproteins. A slight increase in OCI-My5 cells in the sub-G₀ and G₂/Mphase was observed when treated with anti-CD138-mutIFNα.

In contrast MM144 and H929 showed a marked accumulation of cells inG₂/M, with H929 also having an accumulation of cells with sub-G₀ contentof DNA (apoptotic cells). To test if HMCLs were undergoing apoptosis asa result of treatment, cells were stained with Alexa Fluor 488 labeledAnnexin V and PI and analyzed by flow cytometry (FIG. 3C). An inductionof apoptosis was observed after treatment for all of the cell linesexcept OCI-My 5; IFNα or the fusion proteins did not induce apoptosis inthis cell line at even higher concentration of 1 nM (data not shown). Inaddition, a relatively high level of basal apoptosis was observed forOCI-My 5 in multiple experiments. In contrast, apoptosis was induced inall of the other HMCLs. 8226/S and ANBL-6 showed a similar level ofapoptosis in response to all three treatments. On the other hand, U266and H929 had similar levels of apoptosis in response to IFNα andanti-CD138-IFNα but showed a significantly greater level of apoptosis inresponse to anti-CD138-mutIFNα; MM144 also showed some improvement withanti-CD138-mutIFNα. These data are summarized in Table 4. Interestingly,the cytoreductive effects of IFNα appear to involve various mechanismssuch as cell cycle arrest, apoptosis and senescence, and for some celllines, more than one pathway appears to be at work.

TABLE 4 The differential effects of IFNα and fusion proteins against MMcell lines. Construct 8226/S ANBL-6 H-929 MM144 U266 OCI-My 5 Cell cyclearrest IFNα − − + ++ − − Anti-CD138-IFNα − − + ++ − − Anti-CD138-mutIFNα− − ++ + − + Apoptosis IFNα +/− + + + + − Anti-CD138-IFNα +/− + + + + −Anti-CD138-mutIFNα +/− + ++ + ++ − Senescence IFNα + − + +/− − −Anti-CD138-IFNα + − + +/− − − Anti-CD138-mutIFNα + − + +/− − +

Although there was some change to the cell cycle when OCI-My 5 cellswere treated with anti-CD138-mutIFNα, apoptosis was not observed. Inaddition, treatment with IFNα and anti-CD138-IFNα did not causeapoptosis or blocks to cell cycle progression in OCI-My 5 cells. Toconfirm that IFNα and the fusion proteins have a cytoreductive effect,we tested the proliferative status of the cells after treatment.³[H]-thymidine incorporation was measured after 3 days of treatment withvarying concentrations of IFNα, anti-CD138, anti-CD138-IFNα, oranti-CD138-mutIFNα (FIG. 4A). A decrease in proliferation was observedin OCI-My 5 cells, with the fusion proteins having a greater effect thanIFNα alone. Furthermore, the higher affinity anti-CD138-mutIFNα caused agreater effect than the wild type anti-CD138-IFNα. Anti-CD138 IgG didnot decrease the ability of the cells to proliferate although somedecrease was seen at the highest concentrations.

Another possibility for the decrease in cell viability and proliferationis oncogene-induced senescence (OIS). OIS is thought to be part of asafeguard mechanism to prevent abnormal cells from further expansion.Cells in senescence have been shown to be metabolically active in vitrobut do not divide. We examined if IFNα and/or the fusion proteins couldinduce senescence in OCI-My 5 cells. One common marker for the detectionof senescence is β-gal activity at pH 6, which is a barometer forincreased lysosomal content of senescent cells. OCI-My 5 cells weretreated for 3, 4, 6 or 7 days with 1 nM of IFNα, anti-CD138,anti-CD138-IFNα, or anti-CD138-mutIFNα and β-gal activity detected byflow cytometry. At all time points, there was little or no increase inβ-gal activity following treatment with IFNα, anti-CD138 IgG, oranti-CD138-IFNα. However, there was a large increase in β-gal activityin cells treated with anti-CD138-mutIFNα. A representative experiment at3 days is shown in FIG. 4B. Thus induction of senescence appears toaccount at least in part for the reduced proliferation observedfollowing treatment with anti-CD138-mutIFNα. These data suggest that theinduction of senescence in this HMCL requires the higher affinitymutIFNα and senescence does not seem to explain the inhibition ofproliferation seen with IFNα, or anti-CD138-IFNα.

Taken together, these data indicate that although IFNα and the fusionproteins have cytoreductive activity against many HMCLs, the cell lineshave differential responses against this cytokine. These differencesunderscore the fact that HMCLs and MM cells in vivo are heterogeneous innature.

Fusion Proteins are Effective Against Primary MM Cells from Patients

To determine if the fusion proteins are effective against primarytumors, cells were isolated from multiple myeloma patients. Cells weretreated with 25 nM of unfused anti-CD138, fused anti-CD138-IFNα oranti-CD138-mut IFNα for 3 days and the percentage of viable cellscompared to DMSO-treated control (arbitrarily designated as 100%) wasdetermined. The assays used were trypan blue exclusion or MTT assay.

There was some variability in the response of the cells from fivepatients. Generally, the unfused IgG had little effect, while treatmentwith IFNα fusion proteins, especially the mutant, caused significantdecreases in cell viability (FIG. 5, panel A). To determine the timecourse for the effects of the fusion proteins, cells from six patientswere treated for 24, 48 and 72 h and the percentage of viable cells wasdetermined. Although not much difference was apparent after 24 h oftreatment, 25 nM of anti-CD138-mutIFNα was more potent in cytoreductiveeffects than 100 nM of anti-CD138-mutIFNα (FIG. 5, panel B).

Fusion Proteins are Effective in a Murine Xenograft Model of MM

To determine if the fusion proteins would be able to provide protectionagainst MM in vivo, we used a murine xenograft models of MM using OCI-My5 cells or U266 cells. SCID mice were injected subcutaneously with 1×10⁷OCI-My 5 cells and treated on days 14, 16 and 18 with 100 μg ofanti-CD138-IFNα or anti-CD138-mutIFNα. Treatment with PBS, anti-CD138,and untargeted anti-DNS-IFNα and anti-CD20-mutIFNα served as controls.Survival was monitored as well as the size of the tumors. Unfusedanti-CD138 and untargeted anti-DNS-IFN increased survival slightly ascompared to PBS control. Treatment with the targeted anti-CD138-IFNαresulted in a significant increase in survival (p≤0.001 compared toPBS), but an even greater effect was observed with anti-CD138-mutIFNα(p=0.0004 compared to anti-CD138-IFNα).

To determine the in vivo efficacy of fusion protein treatment againstthe U266 tumor, NSG mice were injected subcutaneously with 1×10⁷ U226cells and treated on days 14, 16 and 18 as indicated by the black arrowswith 100 μg of the indicated proteins. One group was treated twoadditional times with anti-CD138-mutIFNα on days 25, 32 and 63 asindicated by the grey arrows. Eight mice were treated for each groupexcept 6 mice for the group that received six treatments withanti-CD138-mutIFNα. Survival was monitored as well as the size of thetumors. Targeted anti-CD138-IFNα was much more effective than untargetedanti-DNS-IFNα in protecting against tumor growth. Moreover, targetedanti-CD138-mutIFNα was even more effective than anti-CD138-IFNα. Whenmice were treated with two additional doses of 100 μg ofanti-CD138-mutIFNα on days 25 and 32, tumor growth was delayed withpalpable tumors not observed until day 60. At day 63 mice were treatedwith an additional 100 μg of anti-CD138-mutIFNα which served to delaybut not completely prevent tumor growth. However, it can be speculatedthat treatment with additional quantities of fusion protein may havesucceeded in completely inhibiting tumor growth.

Discussion

IFNα therapy has been used for treatment of MM, but disagreement existsas to its efficacy. However, meta-analysis of 17 trials among 2333patients who received IFN-chemotherapy induction treatment orchemotherapy alone showed significantly superior outcomes in IFN treatedpatients for relapse-free and overall survival; similarly, meta-analysesof maintenance treatments also showed outcomes significantly better inthe IFN treatment arms than in untreated controls (Fritz and Ludwig(2000) Ann. Oncol., 11: 1427-1436), underscoring the fact that IFNα canbe an effective therapeutic against MM. However the systemic toxicityassociated with IFNα treatment as well as its short in vivo half-lifehave limited the clinical efficacy of IFN treatment. Our approach tocircumventing these problems is to fuse IFNα to anti-CD138 IgG in orderto increase its half-life and by targeting, deliver an effective dose ofIFNα to the tumor site without systemic toxicity.

MM is characterized by significant heterogeneity. Our studies as well asothers (Gomez-Benito et al. (2005) FEBS Lett., 579: 6217-6122;Gomez-Benito et al. (2007) Cell Signal 19: 844-854) showed that not allMMs are responsive to treatment with IFNα. However, for those HMCLs thatare sensitive, targeting via the anti-CD138 antibody portion improvedthe efficacy of IFNα against MM, and in some cases, anti-CD138-mutIFNα,which has a higher affinity for IFNAR, was more effective thananti-CD138-IFNα. However, the responsiveness of MM cell lines to IFNαdoes not appear to always correlate with the level of expression ofIFNAR (Gomez-Benito et al. (2005) FEBS Lett., 579: 6217-6122). Ouranalysis of HMCLs revealed that different cell lines exhibit differentresponses. Consistent with previous studies (Arora and Jelinek (1998) J.Biol. Chem., 273: 11799-11805; Minami et al. (2000) Exp. Hemat., 28:244-255; Chen et al. (2001) Blood, 98: 2183-2192; Crowder et al. (2005)Blood, 105: 1280-1287; Gomez-Benito et al. (2005) FEBS Lett., 579:6217-6122; Arulampalam et al. (2011) Exp. Cell Res., 317: 9-19), themechanism of action of IFNα against MM was found to include apoptosis,blockage in cell cycle progression and senescence.

Following treatment H929 and MM144 cell lines both showed dramaticchanges to cell cycle progression and were blocked at G2/M, with H929also having a significant number of cells in the sub-G0 phase. Both alsounderwent apoptosis. Our results were consistent with previous studiesthat showed that MM144 is blocked in G2 when treated with IFNα. H929contains a 4p16::14q32 translocation (overexpresses FGFR3 and MMSET)while MM144 contains a 14q32::16q23 translocation (overexpresses c-maf).Thus, although the two cell lines have a similar response to treatment,they contain different genetic abnormalities. The stop in cell cycleprogression is different from what has been reported for Daudi cellstreated with IFNα, which show a stop at G1 (Subramanian et al. (1997) J.Biol. Chem., 272: 14713-14720).

In contrast, 8226/S, ANBL-6 and U266 did not display changes in the cellcycle, but did undergo apoptosis in response to IFNα alone and to IFNαfusion treatment; ANBL-6 is IL-6 dependent. Both 8226/S and ANBL-6 arehyperdiploid with a 14q32::16q23 translocation. On the other hand, U266is hypodiploid with a 11q13 insertion and overexpresses cyclin D1. U266has previously been shown to have biallelic deletion of RB gene and doesnot express pRB (Dao et al. (1994) Leukemia, UK 8: 1280; Corradini etal. (1994) Leukemia, 8: 758).

Unlike the other HMCLs analyzed, the OCI-My 5 cell line did not undergoapoptosis, become blocked in cell cycle progression or undergosenescence when treated with IFNα or anti-CD138-IFNα. However, changesto the cell cycle and induction of senescence were observed when cellswere treated with anti-CD138-mutIFNα, suggesting that the higheraffinity of mutIFNα for IFNAR is necessary to affect this cell line. Thereason for the cytoreductive effects of anti-CD138-IFNα against OCI-My 5cells as detected by MTS assays (FIG. 3) is not clear.

The two main pathways of oncogene-induced senescence are p16INK4a-RB andARF-p53, which are involved in the execution of proliferative arrest.IFNα has been shown to induce senescence in endothelial cells (Pammer etal. (2006) Lab. Invest., 86: 997-1007); however, no studies have yetreported IFNα-induced senescence in MM.

There did not appear to be a correlation between the type of geneticabnormality and their responsiveness to IFNα fusion treatment. Forexample, the four cell lines with the 14q32::16q23 translocation andoverexpression of c-maf (OCI-My 5, 8226/S, H929 and ANBL-6) showedapoptosis, cell cycle, or senescence in response to IFNα fusion proteintreatment.

These findings suggest that the variable responses of the differentHMCLs may be attributed to the heterogeneity, some of which is outlinedin Table 4, in the molecular pathogenesis of MM.

In addition to in vitro data using HMCLs, we show that the targeted IFNαfusion proteins are effective against primary cells from MM patients andin a murine xenograft model. These data suggest that targeting of IFNαvia the anti-CD138 moiety may be an effective strategy in the treatmentof MM. In primary cells (and in the mouse model?), the higher affinityanti-CD138-mutIFNα fusion protein was more effective than being moreeffective than wildtype anti-CD138-IFNα. Although the treatments wereeffective in prolonging survival in mice, the fusion proteins may proveto be even more effective in the treatment of human MM patients becauseof immunomodulatory activities of IFNα as well as the IgG Fc region. Itis conceivable that in addition to the direct effects of IFNα, thefusion proteins may also activate ADCC and CDC, further contributing tocancer cell killing in patients.

Signaling

pRb protein is involved in regulating progression through G1 into Sphase; however, the HMCLs with blocks in cell cycle progression werearrested at G2/M and some at sub-G0.

Example 2 Anti-CD138-Interferon α2 Fusion Proteins are Effective InVitro and In Vivo Against Multiple Myeloma

Translational Relevance

It is estimated that in the United States 22,350 individuals will bediagnosed with and 10,700 will die of myeloma in 2013. Although muchprogress has been made in the treatment of myeloma, most treatedindividuals relapse, and myeloma remains an incurable disease.Therefore, innovative therapeutic approaches are desperately needed. IFNtreatment has shown some efficacy for the treatment of myeloma, but theassociated toxicities have limited its efficacy. We propose a novelapproach in which IFN is targeted to myeloma cells expressing CD138using anti-CD138-IFN fusion proteins. We believe that the targeted IFNis effective in killing myeloma cells and in eliciting a tumor specificimmune response.

Purpose:

Multiple myeloma, a plasma cell malignancy characterized by a highdegree of heterogeneity, is the second most prevalent hematologicmalignancy in the US. Although much effort has been made trying tounderstand the etiology and the complexities of this disease with thehope of developing effective therapies, multiple myeloma remainsincurable at this time. Because of their anti-proliferative andpro-apoptotic activities, IFNs have been used to treat variousmalignancies including multiple myeloma. Although some success has beenobserved, the inherent toxicities of IFNs limit their efficacy.

Experimental Design:

To address this problem, we produced anti-CD138 antibody fusion proteinscontaining either IFNα2 or a mutant IFNα2 (IFNα2^(YNS)) with the goal oftargeting IFN to CD138 expressing cells, thereby achieving effective IFNconcentrations at the site of the tumor in the absence of toxicity.

Results:

The fusion proteins inhibited the proliferation of a variety of celllines that represent different molecular and biological multiple myelomasubtypes. Depending on the cell line, the interference with growthfollowing treatment with the fusion proteins included the induction ofapoptosis, blocks in cell cycle progression and/or senescence. Inaddition, the fusion proteins were effective against primary cells frommultiple myeloma patients, and treatment with fusion proteins prolongedsurvival in two different xenograft models. These studies suggest thatIFNα antibody fusion proteins may be effective novel therapeutics forthe treatment of multiple myeloma.

Introduction

Multiple myeloma is a disease characterized by an excess of malignantplasma cells in the bone marrow. Accumulation and proliferation ofmalignant myeloma cells result in disruption of normal hematopoiesis andchanges to bone marrow vascularization and bone physiology. Analyses ofpatient myeloma cells and human myeloma cell lines (HMCLs) have revealedthe extensive molecular heterogeneity of this disease (Carrasco et al.(2006) Cancer Cell 9: 313-325; Drexler et al. (2000) Leukemia, 14:777-782; Lombardi et al. (2006) Genes Chromosomes Cancer, 46: 226-238;Moreaux et al. (2011) Haematologica, 96: 574-582). The survival rate formultiple myeloma is 7-8 years when patients are treated with drugs suchas proteasome inhibitor bortezomib, or thalidomide and lenalidomide,which target myeloma cells in the bone marrow microenvironment (Kumar etal. (2008) Blood, 111: 2516-2520). Currently there is no cure formultiple myeloma.

Besides their anti-viral and immunostimulatory activities, IFNs haveanti-proliferative activity and can induce apoptosis in hematologicalmalignancies and solid tumors (Borden et al. (2000) Semin. Cancer Biol.,10: 125-144; Borden et al. (2007) Nat. Rev. Drug Discov., 6: 975-990).Many studies have shown that type I IFNs, which were the firstrecombinant proteins used in the treatment of cancer, may be highlyeffective against a variety of tumor cell targets (reviewed in Borden etal. (2007) Nat. Rev. Drug Discov., 6: 975-990). Both IFNα and IFNβ bindto the same receptor composed of two transmembrane proteins, IFNAR1 andIFNAR2. However, IFNβ has a 20- to 50-fold greater affinity for IFNAR1than IFNα2, and this greater affinity has been shown to correlate with asignificantly higher anti-proliferative activity against somemalignancies (Jaitin et al. (2006) Mol. Cell Biol., 26: 1888-1897; Kalieet al. (2007) J. Biol. Chem., 282: 11602-11611). However, theeffectiveness of type I IFNs for cancer therapy has been overshadowed bytheir associated side effects when used at high doses (Weiss (1998)Semin. Oncol., 25: 9-13) and a short half-life of only 1 hour(Peleg-Shulman et al. (2004) J. Med. Chem., 47: 4897-4904). Previously,we produced an anti-CD20-IFNα2 fusion protein and showed that targetingto CD20 expressed on tumor cells resulted in potent anti-proliferativeand pro-apoptotic effects on human B cell lymphoma cell lines in vitroand in a murine lymphoma model (Xuan et al. (2010) Blood, 115:2864-2871). We have also shown that fusion of IFNα or IFNβ to IgGincreased the half-life to 8 hours (Huang et al. (2007) J. Immunol.,179: 6881-6888; Trinh et al. (2013) J. Immunother., 36: 305-318).

To determine if this approach would also be effective against multiplemyeloma, we constructed fusions of anti-CD138 with IFNα2 andIFNα2^(YNS), a high affinity IFNα2 mutant (Jaitin et al. (2006) Mol.Cell Biol., 26: 1888-1897; Kalie et al. (2007) J. Biol. Chem., 282:11602-11611). CD138, also known as syndecan-1, is a heparan sulfateproteoglycan that is highly expressed on HMCLs and malignant plasmacells in peripheral blood and in the bone marrow in patients (Chilosi etal. (1999) Mod. Pathol., 12: 1101; Ridley et al. (1993) Blood 81:767-774; Wijdenes et al. (1996) Br. J. Haematol., 94: 318-323).Treatment with IFNα fusion proteins resulted in the induction ofapoptosis, blockage in cell cycle and/or senescence in different HMCLs.In addition, the fusion proteins were effective against primary patientcells and in vivo against multiple myeloma tumors in murine models.

Materials and Methods

Cells

HMCLs were obtained through the generous gift of Dr. W. Michael Kuehland Dr. Diane Jelinek. Their authenticity was confirmed by anti-CD138staining. Primary cells were obtained after informed consent andapproved by the institutional medical ethical committee. HMCLs werecultured in RPMI 1640 (Invitrogen) supplemented with 5% fetal calf serum(FCS; Atlanta Biologics). ANBL-6 cells were cultured as described withthe addition of 2 ng/mL of IL-6. Chinese Hamster Ovary (CHO) cells werecultured in Iscove's Modified Dulbecco's Medium (IMDM; Invitrogen)supplemented with 5% FCS.

Construction of Expression Vectors, Protein Production and Purification

The anti-CD138 heavy and light chain variable (V) region amino acidsequences were obtained from US Patent Application No: 2009/0175863. Thesequences were used to construct expression vectors as described inSupplemental methods. To construct the DNA vector for the expression ofanti-CD138-IFNα2^(YNS), nested PCR was used to introduce three aminoacid mutations (H57Y, E58N, and Q61S) as described in Supplementalmethods.

Expression vectors were stably transfected into CHO cells to producefusion proteins and purified using protein A affinity chromatography asdescribed in Supplemental methods.

MTS and Apoptosis Assays

HMCLs were treated with IFNα2, IFNβ, anti-CD138, anti-CD20-IFNα2,anti-CD20-IFNα2^(YNS), anti-CD138-IFNα2 or anti-CD138-IFNα2^(YNS).Metabolic activity was determined using MTS assay as described inSupplemental methods. Apoptosis was measured by staining cells withAlexa Fluor 488-labeled Annexin V and propidium iodide (PI) as describedin Supplemental methods.

Cell Cycle Analysis

Cells were treated with 500 pM of IFNα2, IFNβ, anti-CD138-IFNα2, oranti-CD138-IFNα2^(YNS) for 4 days at 37° C. Cells were incubated with 1ml of hypotonic DNA staining buffer (1 mg/ml sodium citrate, 100 μg/mlPI, 20 μg/ml RNase A, and 0.3% Triton X-100) for 30-60 minutes at 4° C.in the dark. Cells were analyzed by flow cytometry and cell cycleanalysis performed using FlowJo software with the Watson Model.

Detection of ppRb and IRF-4

Cells were treated for 48 hours with 1 nM IFNα2, IFNβ, anti-CD138-IFNα2,or anti-CD138-IFNα2^(YNS) and analyzed by Western blotting usinganti-IRF-4 or anti-ppRb Ser807/811 as described in Supplemental methods.

Assays for Replicative Senescence

Cells were treated with IFNα2, anti-CD138, anti-CD138-IFNα2, oranti-CD138-IFNα2^(YNS) for 3 days. Senescence induced β-galactosidase(β-gal) activity was detected as described previously (Debacq-Chainiauxet al. (2009) Nat. Protoc., 4: 1798-1806) and in Supplemental methods.To detect Ki-67 protein, cells were stained and analyzed by flowcytometry as described in Supplemental methods.

In Vivo Anti-Tumor Activity Against OCI-My5 and U266 Cells

Six-to-eight week old female scid mice were used to establish OCI-My5tumors. Mice were inoculated subcutaneously with 1×10⁷ cells at the baseof the tail. Mice were treated intravenously with PBS or 100 μg ofanti-CD138, anti-dansyl (DNS)-IFNα2, anti-CD138-IFNα2,anti-CD20-IFNα2^(YNS), anti-CD138-IFNα2^(YNS) on days 14, 16, 18 posttumor challenge. Each group consisted of eight mice. Bidirectional tumorgrowth was measured throughout the experiment, and mice were sacrificedwhen tumors reached 1.5 cm as per institutional guidelines. For U266tumors, experiments were carried out as described above except thatNOD-scid IL2rγ^(null) (NSG) mice were used. All animal studies wereperformed in compliance with the U.S. Department of Health and HumanServices Guide for the Care and Use of Laboratory Animals and wereapproved by the UCLA Animal Research Committee.

Treatment of Primary Myeloma Cells from Patients

Patients with active myeloma were biopsied while off therapy and myelomacells isolated by negative antibody selection to >95% purity. Cells wereincubated with 25 or 100 nM of anti-CD138, anti-CD138-IFNα2 oranti-CD138-IFNα2^(YNS) for 72 hours. Percent viable cell recovery wasdetermined by trypan blue staining, with untreated control cellsdesignated as 100%.

Results

Production and Characterization of Anti-CD138-IFNα2 andAnti-CD138-IFNα2^(YNS) Fusion Proteins

The aim of this study was to test the effectiveness of using antibodiesspecific for CD138 to target IFN to multiple myeloma cells. The approachwas to genetically fuse IFN to the end of the C_(H)3 domain of humanIgG1 containing the V regions from the anti-CD138 antibody B-B4(Wijdenes et al. (1996) Br. J. Haematol., 94: 318-323). We elected totarget IFNα2, which has been used successfully in the treatment ofmultiple myeloma in the clinic. IFNβ binds to the same receptor as IFNαbut has a greater affinity and activity than IFNα2. However, we foundthat fusion of human IFNβ to IgG resulted in a >100-fold decrease inactivity (data not shown). As an alternative approach, we also electedto target IFNα2 containing mutations at three positions, H57Y/E58N/Q61S(anti-CD138-IFNα2^(YNS)). These mutations result in a 60-fold increasedaffinity for IFNAR1 and a large increase in anti-proliferative activitycompared to wildtype IFNα2 (Kalie et al. (2007) J. Biol. Chem., 282:11602-11611). Anti-CD138 IgG1 either unfused or fused to human IFNα2 orIFNα2^(YNS) (FIG. 13A) was expressed in stable CHO transfectants.Purified proteins were characterized with respect to their size andassembly status and were found to possess heavy (H) and light (L) chainsof the appropriate molecular weight and assemble into complete H₂L₂molecules (FIG. 13B) that bound antigen (data not shown).

IFNα2 Fusion Proteins Inhibit the Growth of HMCLs

Multiple myeloma is characterized by biological and geneticheterogeneity (Carrasco et al. (2006) Cancer Cell 9: 313-325; Drexler etal. (2000) Leukemia, 14: 777-782; Lombardi et al. (2006) GenesChromosomes Cancer, 46: 226-238; Moreaux et al. (2011) Haematologica,96: 574-582) and often differ in their responses to various drugs andtreatments. Therefore, we assembled a panel of thirteen HMCLs (XG-1,XG-2, OPM-1, OPM-2, S6B45, delta 47, RPMI 8226, 8226/Dox40, U266,OCI-My5, ANBL-6, NCI-H929 and MM1-144) to investigate their response toIFNα and the fusion proteins. The panel includes both hyperdiploid andnonhyperdiploid cells with various chromosomal translocations (Drexleret al. (2000) Leukemia, 14: 777-782; Moreaux et al. (2011)Haematologica, 96: 574-582; Gabrea et al. (2008) Genes ChromosomesCancer, 47: 573-590). All of the HMCLs were bound by anti-CD138 IgG,indicating that they express CD138 (data not shown).

To determine if the HMCLs were sensitive to treatment and if targetedIFNα2 was more effective, cells were incubated with varyingconcentrations of fusion proteins for 3 days and metabolic activityassessed by MTS assay. Anti-CD138-IFNα2 was used to target IFNα whileanti-CD20-IFNα2 was used as an untargeted control since HMCLs do notexpress CD20. HMCLs showed different responses to treatment. Under theseconditions both fusion proteins had little or no effect on XG-1, XG-2,OPM-1, OPM-2, and delta 47 while some effect was observed only at highconcentrations for S6B45, RPMI 8226, and 8226/Dox40 (data not shown).However, the targeted anti-CD138-IFNα2 was >10-fold more effective thanuntargeted anti-CD20-IFNα2 for U266, OCI-My5, ANBL-6, NCI-H929 andMM1-144 (FIG. 14, panel A). Therefore, the IFNα2 fusion protein that istargeted to an antigen present on myeloma cells appeared to be moreeffective than an untargeted fusion protein against a number of HMCLsrepresenting multiple myeloma with various molecular abnormalities.

In a different experiment, anti-CD138-IFNα2 fusion protein was comparedto equimolar concentrations of anti-CD138 alone and recombinant IFNα2alone. Anti-CD138-IFNα2 was more effective than recombinant IFNα2 ininhibiting the growth of OCI-My5, ANBL-6, and MM1-144, but not NCI-H929.Comparison of IC₅₀ between anti-CD138-IFNα2 and IFNα2 calculated fromthe data shown in FIG. 14, panel B using Prism software confirmed thatCD138-IFNα2 was more effective than IFNα2 in inhibiting the growth ofU266 (8.3×10⁻⁵ versus 1.2×10⁻² pM), OCI-My5 (8.4×10⁻⁴ versus 0.14 pM),ANBL-6 (1.1×10⁻⁴ versus 2.6×10⁻³ pM), and MM1-144 (4.2×10⁻⁴ versus8.0×10⁻² pM). The mutant fusion proteins were compared to recombinantIFNβ. We found that IFNβ, anti-CD20-IFNα2^(YNS) andanti-CD138-IFNα2^(YNS) showed similar ability to inhibit the growth ofOCI-My5, ANBL-6, NCI-H929 and MM1-144 (FIG. 14, panel C). However, forU266 anti-CD138-IFNα2^(YNS) was more effective in growth inhibition(IC₅₀=2.1×10⁻⁷ pM) than IFNβ (2.8×10⁻⁴ pM) or anti-CD20-IFNα2^(YNS)(2.4×10⁻⁴ pM). Unfused anti-CD138 had no effect against any of the fivecell lines (FIG. 14, panel B).

To determine if apoptosis was being induced in the HMCLs, cells weretreated for 3 days at high (500 pM) or low (1 pM or 5 pM for OCI-My5)concentrations of IFNα2 and fusion proteins, stained using Alexa Fluor488-labeled Annexin V and PI and examined by flow cytometry. Whentreated at 500 pM, IFNα2 and the fusion proteins caused apoptosis in allHMCLs (FIG. 15A). For U266 and NCI-H929, hgher levels of apoptosis wereobserved with anti-CD138-IFNα2^(YNS) than with IFNα2 oranti-CD138-IFNα2. In contrast, different results were observed at lowconcentrations. At low concentrations, IFNα2 and the fusion proteins didnot cause apoptosis in ANBL-6, NCI-H929, and MM1-144 (FIG. 15B). Howeverat low concentrations, anti-CD138-IFNα2 and anti-CD138-IFNα2^(YNS) wereable to induce apoptosis in U266 and OCI-My5 while IFNα2 had no effect.These data suggest that although the efficacy appeared to be similar athigh doses, at low doses, targeted fusion protein can have a greatereffect than IFNα2 alone as in the case of U266 and OCI-My5. IFNβ andanti-CD138-IFNα2^(YNS) induced similar levels of apoptosis in U266,NCI-H929 and MM1-144 at both 500 pM and 1 pM concentrations (data notshown).

IFNα2 and Fusion Proteins Can Induce Alterations in Cell CycleProgression and Senescence in Some HMCLs

HMCLs were also analyzed for changes in cell cycle progression followingtreatment with IFNα, IFNβ or fusion proteins for 4 days. All treatmentsresulted in increases in the percentage of dead cells with sub-G₀/G₁ DNAcontent (FIG. 16A and Table 5). The most profound cell cycle changeswere observed for NCI-H929 and MM1-144, which showed large increases inthe percentage of cells in G₂/M with concomitant decreases in cells inG₁. For the other HMCLs, the changes were more subtle. ANBL-6 showed adecrease in cells in S phase while U266 and OCI-My5 showed only smallcell cycle changes following treatment. In all cases, comparable changeswere seen following treatment with IFNα2, IFNβ, anti-CD138-IFNα2 andanti-CD138-IFNα2^(YNS).

TABLE 5 The percentages of live cells in different phases of the cellcycle after treatment with the indicated proteins are shown. % G₁ % S %G₂/M >G₂ Sub G₀/G₁ U266 Untreated 42.6 28.5 17.4 10.3 5.2 IFNα2 52.523.6 14.3 5.7 14.0 Anti-CD138-IFNα2 53.9 21.4 15.1 5.5 13.2Anti-CD138-IFNα2^(YNS) 58.5 13.1 20.2 6.4 15.9 IFNβ 58.7 12.4 20.1 7.117.0 OCI-My5 Untreated 37.4 41.7 19.0 0.1 9.4 IFNα2 30.1 45.6 19.2 1.219.0 Anti-CD138-IFNα2 25.6 49.0 20.5 1.5 17.9 Anti-CD138-IFNα2^(YNS)23.8 50.3 20.5 0.0 24.2 IFNβ 21.8 54.2 19.0 0.0 29.5 ANBL-6 Untreated51.8 25.3 10.1 4.6 11.3 IFNα2 64.8 14.3 17.0 0.0 19.8 Anti-CD138-IFNα263.6 14.0 18.0 0.0 19.6 Anti-CD138-IFNα2^(YNS) 64.1 14.7 17.7 0.0 10.8IFNβ 60.5 15.4 18.8 0.0 26.1 NCI-H929 Untreated 62.1 21.4 11.9 2.7 1.8IFNα2 41.5 21.7 25.4 7.6 21.2 Anti-CD138-IFNα2 41.9 21.0 24.3 8.2 18.9Anti-CD138-IFNα2^(YNS) 43.0 18.0 26.6 8.0 30.4 IFNβ 41.2 22.1 23.4 7.837.4 MM1-144 Untreated 48.5 29.4 13.9 7.0 5.1 IFNα2 21.0 31.5 34.2 11.610.3 Anti-CD138-IFNα2 22.6 29.9 34.3 11.2 11.3 Anti-CD138-IFNα2^(YNS)27.7 30.0 30.7 9.4 11.9 IFNβ 29.0 33.7 26.7 8.6 11.7

Phosphorylated retinoblastoma protein (ppRB), which is regulated byinteractions between cyclin D and cyclin-dependent kinases, is anindicator of cell cycle progression. Since cell cycle progression wasblocked in some HMCLs, we determined the levels of ppRB by Westernblotting (FIG. 16B). We did not detect any ppRB in U266, which has abiallelic deletion of the RB gene (Corradini et al. (1994) Leukemia, 8:758; Dao et al. (1994) Leukemia, 8: 1280; Juge-Morineau et al. (1995)Br. J. Haematol., 91: 664-667). For NCI-H929, MM1-144 and OCI-My5, ppRblevels were decreased following treatment with IFNα2, anti-CD138-IFNα2and anti-CD138-IFNα2^(YNS), consistent with these proteins having anegative effect on cell division. Similar results were obtained withANBL-6 (data not shown).

Cell cycle arrest may be an indicator of replicative senescence.Although this arrest has been described to occur in G₀/G₁ (reviewed inKong et al. (2011) J. Aging Res., 2011: 963172), G₂ arrest has also beenreported (Mao et al. (2012) Aging, 4: 431; Olsen et al. (2002) Oncogene,21: 6328; Wada et al. (2004) Nat. Cell Biol., 6: 215-226; Zhu et al.(1998) Genes Dev., 12: 2997-3007). IFNα2 has been reported to inducesenescence in endothelial cells (Pammer et al. (2006) Lab. Invest. 86:997-1007), but no such activity has yet been reported in myeloma. Onemarker for senescence is β-gal activity at pH 6, which is a barometer ofincreased lysosomal content in the cells undergoing senescence. To assayfor senescence, HMCLs were treated for 3 days with IFNα2, anti-CD138,anti-CD138-IFNα2, or anti-CD138-IFNα2^(YNS) and β-gal activity detectedby flow cytometry (FIG. 16C). NCI-H929 and MM1-144 showed increases inβ-gal activity following treatment with IFNα2, anti-CD138-IFNα2 andCD138-IFNα2^(YNS) but not with anti-CD138. In the case of NCI-H929,targeting with anti-CD138-IFNα2 and CD138-IFNα2^(YNS) had a greatereffect than with IFNα2 alone. Interestingly, OCI-My5 showed an increaseonly following treatment with anti-CD138-IFNα2^(YNS). U226 showed noincrease under any conditions; similarly, ANBL-6 also showed noincreases (data not shown).

Ki-67 protein is present during all active phases of the cell cycle butis absent from cells that have ceased dividing, including senescentcells. To quantify the number of non-dividing cells, HMCLs were treatedfor 3 days and Ki-67 expression was determined by flow cytometry. Thepercentage of Ki-67 (non-dividing) cells are shown in FIG. 16D. If cellscease dividing in response to treatment, the percentage of Ki-67 cellsshould increase as compared to untreated cells. Indeed, this was what weobserved for NCI-H929 and MM1-144. Treatment with IFNα2,anti-CD138-IFNα2, and anti-CD138-IFNα2^(YNS) resulted in more Ki-67cells as compared to untreated control. For OCI-My5, an increase inKi-67 cells was observed only with anti-CD138-IFNα2^(YNS), which wasalso the only treatment that resulted in increased β-gal activity (FIG.16C). Thus it appears that IFNα2 can induce senescence in some HMCLs. Inparticular for NCI-H929 and MM1-144, the accumulation of cells in G₂/Mand increases in β-gal activity and Ki-67 cells are evidence that thesecell lines are undergoing senescence in response to IFNα2 and fusionprotein treatment.

Expression of the transcription factor IFN regulatory factor-4 (IRF-4)has been shown to be required for survival of multiple myeloma cellsregardless of their genetic etiology, with even small changes to IRF-4levels resulting in cell death (Shaffer et al. (2008) Nature, 454:226-231). Therefore, we wanted to determine if treatment resulted inchanges to IRF-4 levels. While IRF-4 levels were unchanged in NCI-H929,MM1-144, OCI-My5 and ANBL-6 (data not shown), there was a decrease inIRF-4 expression in U266 after treatment with IFNα2 and fusion proteins(FIG. 16B). The observed decrease was similar when U266 was treated withIFNα2, anti-CD138-IFNα2 and anti-CD138-IFNα2^(YNS). Thus for U266, thefusion proteins interfere with tumor growth at least in part by causinga decrease in IRF-4 expression.

Fusion Proteins are Effective in Murine Xenograft Models of MultipleMyeloma

To determine if the fusion proteins are protective against multiplemyeloma in vivo, we used xenograft models of OCI-My5 and U266 tumors. Inthe OCI-My5 model, tumors were established in scid mice, which lackmature T and B cells. Mice were treated on days 14, 16 and 18. To treatthe mice, we used unfused anti-CD138, an untargeted fusion protein withan irrelevant specificity for the hapten dansyl (DNS; anti-DNS-IFNα2),untargeted anti-CD20-IFNα2^(YNS), or targeted anti-CD138-IFNα2 andanti-CD138-IFNα2^(YNS). We would not expect anti-CD20-IFNα2^(YNS) totarget to any cells in the mice since it is specific for human CD20.Survival and tumor size were monitored (FIG. 17A). The survival ofanti-CD138 treated mice did not differ significantly from PBS treatedmice (p=0.079), consistent with our failure to observe anti-tumoractivity by anti-CD138 in vitro. All of the fusion proteins includingthose that were not targeted to CD138 showed some level of protection.However, targeting resulted in significant improvements to survival withgreater protection observed with anti-CD138-IFNα2 than with untargetedanti-DNS-IFNα2 (p=0.021) and anti-CD138-IFNα2^(YNS) than with untargetedanti-CD20-IFNα2^(YNS) (p=0.0075). In addition, anti-CD138-IFNα2^(YNS)was more protective than anti-CD138-IFNα2 (p<0.0001). Surprisingly, theuntargeted anti-CD20-IFNα2^(YNS) was more effective than the targetedanti-CD138-IFNα2 fusion protein (p=0.0007).

In the U266 tumor model, NSG mice were used. NSG mice are severelyimmunocompromised, lacking mature T and B cells, functional NK cells andare deficient in cytokine signaling. Mice were treated on days 14, 16and 18 as described above. All treatment groups, including anti-CD138and untargeted IFNα2 fusion proteins, showed significant improvements insurvival when compared with the untreated PBS control (p≤0.0003; FIG.17B). However, the targeted anti-CD138-IFNα2 and anti-CD138-IFNα2^(YNS)displayed the highest levels of protection. Althoughanti-CD138-IFNα2^(YNS) was more effective than anti-CD138-IFNα2 for U266in in vitro assays, the two proteins had similar protective effects invivo (p=0.05). Interestingly, anti-CD138 also showed significantprotection when compared with the untargeted anti-DNS-IFNα2 (p<0.0001)and anti-CD20-IFNα2^(YNS) (p=0.0024) in this murine model even though itshowed no growth inhibition activity in vitro (FIG. 14). Untargetedanti-CD20-IFNα2^(YNS) was more effective than untargeted anti-DNS-IFNα2(p=0.001), consistent with its greater IFN activity.

Fusion Proteins are Effective Against Primary Multiple Myeloma Cellsfrom Patients

To determine if the fusion proteins are effective against primarytumors, multiple myeloma patient cells were treated with anti-CD138,anti-CD138-IFNα2 or anti-CD138-IFNα2^(YNS) for 72 hours and thepercentage of viable recovered cells compared to untreated cells wasdetermined by trypan blue exclusion. As expected, untreated primarycells demonstrated no increase in cell number during this time asfreshly obtained primary multiple myeloma cells do not proliferate exvivo. Not surprisingly, there was some variability in response among theseven patients' cells when the proteins were tested at 25 nM (FIG. 18,panel A). Anti-CD138, anti-CD138-IFNα2 and IFNα2 (data not shown) hadlittle effect at this concentration. In contrast, anti-CD138-IFNα2^(YNS)treatment resulted in significant decreases to cell viability whencompared to anti-CD138 (p<0.0001) or to anti-CD138-IFNα2 (p=0.0009).Although anti-CD138-IFNα2 was not effective at 25 nM, at 100 nManti-CD138-IFNα2 was able to significantly reduce cell viability(p=0.0026; FIG. 18, panel B). These data show that although both fusionproteins can affect cell viability, fusion with the higher affinityIFNα2^(YNS) is more effective at lower concentrations against primarypatient cells. In a few primary samples where sufficient cell numberswere available to perform apoptosis assays, fusion proteins appeared toinduce apoptotic death (data not shown).

Discussion

IFNα therapy has been used for treatment of multiple myeloma, butdisagreement exists as to its efficacy. However, meta-analysis of 17trials including 2333 patients who received combinationIFNα-chemotherapy or chemotherapy alone showed significantly superioroutcomes in IFNα treated patients for relapse-free and overall survival;similarly, meta-analyses of maintenance treatments also showedsignificantly better outcomes in the IFNα treatment arms than inuntreated controls (Fritz et al. (2000) Ann. Oncol., 11: 1427-1436),underscoring the fact that IFNα can be an effective therapeutic againstmultiple myeloma. Some of the major problems for IFNα2 therapy aresystemic toxicity and short in vivo half-life. Our approach tocircumventing these problems was to fuse IFNα2 to anti-CD138 IgG1 toincrease its half-life and by targeting, deliver an effective dose ofIFNα2 to the tumor site without systemic toxicity. We have previouslyused this approach successfully in the treatment of human B celllymphoma using an anti-CD20-IFNα2 fusion protein and have shown that itis significantly more effective than anti-CD20 antibody alone or thecombination of anti-CD20 and IFNα2 in mice (Xuan et al. (2010) Blood,115: 2864-2871).

Multiple myeloma is characterized by significant heterogeneity andindeed our studies have shown heterogeneity in the response of differentHMCLs to treatment with IFNα and fusion proteins. Our studies as well asothers (Crowder et al. (2005) Blood., 105: 1280-1287; Gomez-Benito etal. (2005) FEBS Lett., 579: 6217-6222) have shown that not all multiplemyeloma cells are responsive to treatment with IFNa; the responsivenessof HMCLs does not always correlate with the level of IFNAR expression(Gomez-Benito et al. (2005) FEBS Lett., 579: 6217-6222). For HMCLs thatwere sensitive, fusion proteins containing wildtype IFNα2 or mutantIFNα2^(YNS) were comparable to or better than IFNa alone in all of thein vitro assays, suggesting that targeting via anti-CD138 enhancesefficacy. The inhibition of proliferation observed resulted at least inpart from the induction of apoptosis, alterations in cell cycle andsenescence.

Replicative senescence is recognized as a potential mechanism to preventtumorigenesis and cancer progression. In vitro, senescent cells do notdivide but are viable and metabolically active. IFNα has been shown toinduce senescence in endothelial cells (Pammer et al. (2006) Lab.Invest. 86: 997-1007), and IFNβ has been shown to induce senescence inhuman papilloma virus-transformed keratinocytes (Chiantore et al. (2012)PLoS One, 7: e36909), biliary epithelial cells (Sasaki et al. (2008)Free Radic. Res., 42: 625-632), and human fibroblasts (Moiseeva et al.(2006) Mol. Biol. Cell, 17: 1583-1592). However, IFNα-induced senescencein multiple myeloma has not been reported to date. One novel findingfrom our studies is that IFNα2 and fusion proteins can induce cellularsenescence in some HMCLs as indicated by increases insenescence-associated β-gal activity and decreases in Ki-67 levels.These changes were observed in NCI-H929 and MM1-144 cells when treatedwith IFNα2, anti-CD138-IFNα2 and anti-CD138-IFNα2^(YNS), while OCI-My5displayed such changes only when cells were treated withanti-CD138-IFNα2^(YNS). Alternations in cell cycle caused by IFNα havebeen reported (Crowder et al. (2005) Blood., 105: 1280-1287;Gomez-Benito et al. (2005) FEBS Lett., 579: 6217-6222; Arora et al.(1998) J. Biol. Chem., 273: 11799-11805; Arulampalam et al. (2011) Exp.Cell Res., 317: 9-19; Minami et al. (2000) Exp. Hematol., 28: 244-255),and in our study, NCI-H929 and MM1-144 were blocked at the G₂/M phase.Although most studies have found accumulation of senescent cells in G₁,G₂-arrested senescent cells have also been reported (Mao et al. (2012)Aging, 4: 431; Olsen et al. (2002) Oncogene, 21: 6328; Wada et al.(2004) Nat. Cell Biol., 6: 215-226; Zhu et al. (1998) Genes Dev., 12:2997-3007). HMCLs in which senescence was detected also had populationsof apoptotic cells in response to treatment. Since senescent cells arethought to be resistant to apoptosis, it may be that subpopulations ofcells are responding to IFNα2 differentially. Indeed, a growinghypothesis is that low doses of chemotherapeutic drugs induce senescencewhile high doses trigger apoptosis (Rebbaa et al. (2003) Oncogene, 22:2805-2811; Zheng et al. (2004) Cancer Res. 64: 1773-1780). Theobservation of both senescence and apoptosis in these HMCLs may alsoreflect heterogeneity of the uncloned cell lines.

Expression of IRF-4 is associated with many lymphoid malignancies. IRF-4acts as a master regulator of an aberrant, malignancy-specificregulatory network which influences metabolism, membrane biogenesis,cell cycle progression, cell death and transcriptional regulation inmyeloma cells (Verdelli et al. (2009) Hematol. Oncol. 27: 23-30). IRF-4inhibition has been found to be toxic to myeloma cell lines regardlessof the transforming oncogenic mechanism (Shaffer et al. (2008) Nature,454: 226-231). We found that IFNα2, anti-CD138-IFNα2 andanti-CD138-IFNα2^(YNS) treatment results in decreased expression ofIRF-4 in U266 but not in the other HMCLs tested. Thus decreasedexpression of IRF-4 may contribute to the anti-tumor effects seen inU266.

One of the major challenges in translational research is to determine ifin vitro assays are predictive of in vivo outcome. As was observed invitro, targeting improved the efficacy of IFNα in two in vivo modelswith anti-CD138-IFNα2 always more effective than untargetedanti-DNS-IFNα2 and anti-CD138-IFNα2^(YNS) always more effective thatuntargeted anti-CD20-IFNα2^(YNS). However, other interesting andunexpected effects were also observed in vivo. Surprisingly, in theOCI-My5 model the untargeted anti-CD20-IFNα2^(YNS) was more effectivethan the targeted anti-CD138-IFNα (but less effective than targetedanti-CD138-IFNα2^(YNS)) even though in in vitro studies,anti-CD138-IFNα2 was more effective than anti-CD20-IFNα2^(YNS) with IC₅₀of 8.4×10⁻⁴ pM versus 2.3×10⁻³ as calculated from the MTS assay (FIG.14). The superior efficacy of anti-CD138-IFNα2^(YNS) in vitro and invivo and the ability of anti-CD20-IFNα2^(YNS) to provide protection inmice suggest that OCI-My5 may be highly sensitive to IFNα2^(YNS).

Targeting also increased the anti-tumor effects against U266 tumors inNSG mice with anti-CD138-IFNα2 more effective than anti-DNS-IFNα2 andanti-CD138-IFNα2^(YNS) more effective than anti-CD20-IFNα2^(YNS).However, in contrast to what was observed with OCI-My5, the effects ofanti-CD138-IFNα2 and anti-CD138-IFNα2^(YNS) were comparable in the U266in vivo model. This was unexpected given that in vitro,anti-CD138-IFNα2^(YNS) was more effective than anti-CD138-IFNα2 againstU266 cells in the MTS assay and in the apoptosis assay at highconcentration. However, at low concentrations, anti-CD138-IFNα2 andanti-CD138-IFNα2^(YNS) had comparable ability to induce apoptosis (FIG.15B), suggesting that the lower concentration may more accuratelyreflect the in vivo situation. Surprisingly, although anti-CD138 did notdisplay any anti-tumor activity in vitro, anti-CD138 was more effectivethan the untargeted IFNα2 and untargeted IFNα2^(YNS) fusion proteins ininhibiting tumor growth in vivo, suggesting that tumor cell growthinhibition is achieved at least in part through the effector functionsof IgG, which would be observable in vivo but not in vitro. Although NSGmice are severely immunocompromised, they do contain functionalmonocytes and neutrophils (Racki et al. (2010) Transplantation, 89:527), which may be involved in tumor killing via antibody-dependentcell-mediated cytotoxicity (ADCC) (Ravetch and Kinet (1991) Annu. Rev.Immunol., 9: 457-492).

Taken as a whole, our data suggest that targeting of IFNα2 via theanti-CD138 moiety can be an effective strategy in the treatment ofmultiple myeloma. Both anti-CD138-IFNα2 and anti-CD138-IFNα2^(YNS) wereeffective against HMCLs in vitro and were able to prolong survival inmice. The higher affinity anti-CD138-IFNα2^(YNS) showed greater activitythan anti-CD138-IFNα2 against some HMCLs, primary myeloma cells, and inthe OCI-My5 xenograft model, suggesting that IFNs with increasedaffinity may be more effective. The fusion proteins may prove to be evenmore effective in the treatment of human patients since theimmunomodulatory activities of human IFNα2 and effector functions suchas complement-dependent cytotoxcity and ADCC associated with the humanIgG Fc region are not fully functioning in mice. Moreover, fusion ofIFNα2 to anti-CD138 should increase the half-life while decreasing thesystemic cytotoxicity of IFNα2, making for a more effective therapeuticagainst multiple myeloma.

Supplemental Methods

Construction of Expression Vectors

The anti-CD138 heavy (H) and light (L) chain variable (V) region aminoacid sequences were obtained from US Patent Publication No: 2009/0175863(U.S. Ser. No. 12/342,285) also published as PCT Publication No:WO2009080829A1 entitled “Agents targeting CD138 and uses thereof” whichis incorporated herein by reference for the antibody sequences providedhterein.

V_(H) sequence: (SEQ ID NO: 58)MGWSYIILFLVATATGVHSQVQLQQSGSELMMPGASVKISCKATGYTFSNYWIQRPGHGLEWIGEILPGTGRTIYNEKFKGKATFTADISSNTVQMQLSSLTSEDSAVYYCARRDYYGNFYYAMDYWGQGTSVTVSS. V_(L) sequence: (SEQ ID NO: 59)MKSQTQVFIFLLLCVSGAHGDIQMTQSTSSLSASLGDRVTISCSASQGINNYLNWYQQKPDGTVELLIYYTSTLQSGVPSRFSGSGSGTDYSLTISNLEPEDIGTYYCQQYSKLPRTFGGGTKLEIK

The DNA sequence encoding a signal peptide was added 5′ of the H chainand L chain V regions (MGWSYIILFLVATATGVHS (SEQ ID NO:60) andMKSQTQVFIFLLLCVSGAHG (SEQ ID NO:61), respectively) as well as thenucleotide sequence containing a Kozak ribosomal recognition site(5′-GGATATCCACC-3′, SEQ ID NO:62). To facilitate downstream cloning, thesequence 5′-GCTAGCC-3′ (SEQ ID NO:63) was added 3′ of the H chain Vregion, and the sequence 5′-CGTAAGTCGACG-3′(SEQ ID NO:64) was added 3′of the L chain V region. The DNA sequence was synthesized using codonsoptimized for CHO expression (DNA2.0).

The L chain V region flanked by EcoR V and Sal I restriction sites wassequence-verified before cloning into an expression vector containingthe human κ L chain constant region. The H chain V region flanked byEcoR V and Nhe I restriction sites was sequence-verified and cloned intoan expression vector containing the human γl H chain constant region toproduce anti-CD138 IgG1. To produce the fusion protein, the anti-CD138V_(H) was cloned into an expression vector containing the human γl Hchain, a Gly-Ser linker (SGGGGS, SEQ ID NO:8), followed by human IFNα2.This expression vector was named pAH6905.

To construct the DNA vector for the expression ofanti-CD138-IFNα2^(YNS), nested PCR was used to introduce three aminoacid mutations—H57Y, E58N, and Q61S. The first round of PCR was doneusing the forward primer 5′-CGC GGA TCC TGT GAT CTG CCT CAA ACC CAC-3′(SEQ ID NO:65) and reverse primer 5′-CCT CTA GAA TCA TTC CTT ACT TCT TAAACT-3′ (SEQ ID NO:66). The nested PCR was done using forward primer5′-CTC TAC AAT ATG ATC TCA CAG ATC-3′ (SEQ ID NO:67) and reverse primer5′-GAT CTG TGA GAT CAT ATT GTA GAG-3′ (SEQ ID NO:68), which contain themutations to IFNα2. The insert was cloned into pCR2.1-TOPO vector(Invitrogen) and the DNA sequence was verified. The Xba I/BamH Ifragment containing the mutant IFNα2^(YNS) sequence was cloned into anintermediate human γl H chain vector and named pAH11015. The BamH I/AvrII fragment from pAH11015 containing the mutations to IFNα2 was thenused to replace the wildtype IFNα2 sequence from expression vectorpAH6905 (see above).

Protein Production and Purification

Fusion proteins were produced in CHO cells by transfection of H and Lchain expression vectors. Stably transfected cells were selected with 1mM histidinol. To produce IgG and fusion proteins, cells were seededinto roller bottles. At confluency, cells were expanded to 100 mL withIMDM+1% Fetal Clone (Thermo Fisher). The supernatant was removed every2-3 days and replaced with fresh medium. Cell free culture supernatantswere then passed through a protein A-Sepharose 4B fast flow column(Sigma) and the bound protein eluted with 0.1 M citric acid, pH 3.5.Eluted fractions were neutralized immediately with 2 M Tris-HCl pH 8.0.Fractions were run on SDS PAGE gels and stained with Coomassie blue toverify protein purity and integrity. Protein concentrations weredetermined using the BCA assay (Pierce). Anti-CD20-IFNα2,anti-CD20-IFNα2^(YNS) and anti-dansyl (DNS)-IFNα2 used as untargetedcontrol proteins were produced as described previously¹².

MTS Assay to Determine Metabolic Activity

HMCLs were seeded in 96-well plates and incubated with 0.00002 pM-25 nMof IFNα2, IFNβ, anti-CD138, anti-CD20-IFNα2, anti-CD20-IFNα2^(YNS),anti-CD138-IFNα2 or anti-CD138-IFNα2^(YNS) at 37° C. for 3, 4 or 7 days.Metabolic activity was determined using MTS solution (Promega) bymeasuring absorbance at 490 nm using a Synergy HT Multi-DetectionMicroplate Reader (BioTek Instruments Inc.) with untreated cells being100%. GraphPad Prism (GraphPad Software Inc.) was used to analyze databy non-linear regression with the log (inhibitor) versus the responsewith a variable slope. Data are expressed as a percentage of maximummetabolic activity. The experiments were performed in triplicate.

Apoptosis Assay

Cells were incubated with 500 pM of IFNα2, anti-CD138-IFNα2 oranti-CD138-IFNα2^(YNS) for 3 days at 37° C. Cells were stained withAlexa Fluor 488-labeled Annexin V and propidium iodide (PI) using theVybrant Apoptosis Kit #2 (Molecular Probes) as per manufacturer'sinstructions and analyzed by flow cytometry.

Senescence Induced β-Galactosidase Activity

Intracellular β-galactosidase (β-gal) activity was measured as anindicator for replicative senescence. Cells were treated with 1 nM ofanti-CD138, anti-CD138-IFNα2 or anti-CD138-IFNα2^(YNS) for 3 days at 37°C. After treatment, cells were incubated with 100 nM bafilomycin A1(Sigma) for 1 hour to induce lysosomal alkalinization. Then cells wereincubated for 30 minutes with the 33 μM of β-gal substratedodecanoylaminofluorescein di-β-D-galactopyranoside (C₁₂FDG;Invitrogen), which becomes fluorescent after cleavage toC₁₂-fluorescein. After washing twice with cold PBS, cells wereresuspended in cold PBS containing 1 mM phenylethyl thiogalactoside(PETG), a β-gal inhibitor, and analyzed by flow cytometry forC₁₂-fluorescein.

Ki-67 Stain

Cells were treated with 500 pM of IFNα2, anti-CD138-IFNα2 oranti-CD138-IFNα2^(YNS) for 3 days at 37° C. After treatment, cells werefixed with methanol on ice and then incubated with anti-Ki-67 rabbit IgG(Abcam). Cells were then stained with anti-rabbit IgG-FITC (Sigma) andanalyzed by flow cytometry.

Western Blot Analysis of ppRb and IRF-4

Cells were treated for 48 hours with 1 nM IFNα2, anti-CD138,anti-CD138-IFNα2, or anti-CD138-IFNα2^(YNS). Cells were lysed using RIPAbuffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodiumdeoxycholate, 0.1% SDS) containing a protease inhibitor cocktail (RocheApplied Science). The cytosolic fractions were reduced withβ-mercaptoethanol and separated by SDS PAGE. Following transfer tonitrocellulose membrane (Whatman) and blocking, samples were incubatedwith the following primary rabbit antibodies: anti-IRF-4 (Epitomics),anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma), oranti-ppRb Ser807/811 (Santa Cruz Biotechnology). Secondary anti-rabbitIgG-HRP (GE Healthcare) was used and the blots were developed usingenhanced chemiluminescence (ECL; Thermo Scientific). Films were imagedusing a MultiImage™ Light Cabinet (Alpha Innotech Corp.) and analyzedusing NIH Image J. Bands were normalized to a GAPDH loading control andexpressed as percentage band intensity of untreated cells.

Example 3 Additional Data

There are 12 different human IFNαs with different biologic activities(see, e.g., Lavoie et al. (2011) Cytokine 56: 82). We have now made ananti-CD138 fused to human IFNα14 (anti-CD138-IFNα14) and examined itsactivity. The anti-CD138 was fused to the INFα14 by a SGGGGS (SEQ IDNO:8) linker. The amino acid sequence of Human interferon alpha 14(IFNα14) is given in UniProtKB/Swiss-Prot: P01570.3 as:

(SEQ ID NO: 69) 1 MALPFALMMA LVVLSCKSSC SLGCNLSQTH SLNNRRTLML MAQMRRISPFSCLKDRHDFE 61 FPQEEFDGNQ FQKAQAISVL HEMMQQTFNL FSTKNSSAAW DETLLEKFYIELFQQMNDLE 121 ACVIQEVGVE ETPLMNEDSI LAVKKYFQRI TLYLMEKKYS PCAWEVVRAEIMRSLSFSTN 181 LQKRLRRKD

It is noted that MALPFALMMA LVVLSCKSSC SLG (SEQ ID NO:70) constitutesthe hydrophobic leader sequence and was not included in the fusionprotein

FIG. 19 shows the results of an MTS assay following 3 day incubationwith the indicated proteins. Daudi does not express CD138 andnon-targeted anti-CD138-IFNα, anti-CD138-IFNα14 and anti-CD138-mutIFNαshow similar activity.

FIG. 20 shows the results of an MTS assay following 3 day incubationwith the indicated proteins. Targeted anti-CD138-IFNα2 andanti-CD138-IFNα14 show similar activity against the OCI-My5 myeloma.

FIG. 21 shows the results of an MTS assay following 3 day incubationwith the indicated proteins. Anti-CD138-IFNα2 and anti-CD138-IFNα14 showsimilar activity against U266.

The ovarian cancer OVCAR3 expresses low levels of CD138. FIG. 22 showsthat anti-CD138-IFNα14 is more effective than anti-CD138-IFNα2 againstOVCAR3.

The proteasome inhibitor bortezomib (VELCADE® (bortezomib)) is anapproved therapeutic for the treatment of myeloma. We tested forpotential synergy of VELCADE® with anti-CD138 fusion proteins. FIG. 23:Cells were incubated with the indicated treatments for 3 days and thentheir proliferation measured using the MTS assay. For U266 VELCADE wasused at 1.5 nM, anti-CD138-IFN^(YNS) (a.k.a. anti-CD138-mutIFNα) at .5pM and the other antibodies at 1.5 pM. For OCI-My5 VELCADE® was used at1 nM and the antibodies at 1 pM. For the remaining cell lines, VELCADEwas used at 5 nM and the antibodies at 5 pM. The Combination Index (CI)was calculated using Compusyn (see, e.g., Chou and Talalay, (1984) Adv.Enz. Regul. 22: 27-55). Values less than one indicate synergisticinteractions; the smaller the number, the greater the synergy.

FIGS. 24-28: Cells were incubated with the indicated treatments for 3days and then stained with Annexin V and PI and analyzed by flowcytometry. In all cases synergy is seen between VELCADE and the IFNfusion protein in the induction of apoptosis. ANBL-6 and MM1-144 weretreated with 5 pM of antibody or antibody fusion protein and 4 nMVELCADE®. H929 was treated with 5 pM of antibody or antibody fusionprotein and 5 nM VELCADE®. OCI-My5 was treated with 3 pM of antibody orantibody fusion protein and 3 nM VELCADE®. U266 was treated with 3 pM ofantibody or antibody fusion protein and 1 nM VELCADE®.

Lenalidomide, an analog of thalidomide, is FDA approved for thetreatment of multiple myeloma. We also found evidence of synergy betweenanti-CD138-mutIFNα and lenalidomide (FIG. 29).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A chimeric construct comprising an interferonalpha, or a mutant interferon alpha comprising mutations H57Y, E58A, andQ61, attached by a proteolysis-resistant peptide linker that has alength shorter than 15 amino acids to a full-length antibody that bindsCD138, where the amino acid sequence of said linker consists of thesequence SGGGGS (SEQ ID NO:8), and where said chimeric construct iseffective to synergistically enhance the activity of lenalidomide ininducing apoptosis of cancer cells.
 2. The construct of claim 1, whereinsaid construct, when contacted to a cell that expresses or overexpressesCD138, results in the killing or inhibition of growth or proliferationof said cell.
 3. The construct of claim 2, wherein said cell thatexpresses or overexpresses CD138 is a cancer cell.
 4. The construct ofclaim 2, wherein said cell that expresses or overexpresses CD138 is acancer from a cancer selected from the group consisting of multiplemyeloma, ovarian carcinoma, cervical cancer, endometrial cancer, kidneycarcinoma, gall bladder carcinoma, transitional cell bladder carcinoma,gastric cancer, prostate adenocarcinoma, breast cancer, prostate cancer,lung cancer, colon carcinoma, Hodgkin's and non-Hodgkin's lymphoma,chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL),acute myeloblastic leukemia (AML), a solid tissue sarcoma, coloncarcinoma, non-small cell lung carcinoma, squamous cell lung carcinoma,colorectal carcinoma, hepato-carcinoma, pancreatic cancer, and head andneck carcinoma.
 5. The construct of claim 1, wherein said interferon isan interferon-alpha (IFN-α).
 6. The construct of claim 5, wherein saidinterferon is an interferon alpha 2 (IFNα2).
 7. The construct of claim5, wherein said interferon is an interferon alpha 14 (IFNα14).
 8. Theconstruct of claim 1, wherein said interferon is a mutant interferonalpha comprising mutations H57Y, E58A, and Q61S.
 9. The construct ofclaim 1, wherein said antibody comprises the complementarity determiningregions of the B-B4 monoclonal antibody.
 10. The construct of claim 9,wherein said antibody comprises the VH and/or VL domain of the B-B4monoclonal antibody.
 11. The construct of claim 10, wherein saidantibody is the B-B4 monoclonal antibody.
 12. A pharmaceuticalformulation comprising a construct of claim 1 in a pharmaceuticallyacceptable excipient.
 13. The formulation of claim 12, wherein saidformulation further comprises bortezomib and/or lenalidomide.